Dissertations / Theses on the topic 'Antropologia molecolare, DNA, Biologia'

Synthetic biology aims to exploit the development of foundational technologies in the current expansion of biotechnology applications that makes the design and manufacturing of engineered biological systems easier and more reliable. Instrumental to fulfil this vision is the decoupling between design and fabrication. Decoupling is defined as breaking down a complex task into simpler and independent ones such that the resulting work can eventually be recombined to produce a functioning whole. This is in stark contrast with the traditional setting where individual researchers needed to carry out different tasks ranging from DNA design to assembly and quality controls. Thus, decoupling enables a team of complementary experts to leverage individual specializations to achieve better outcomes. By decoupling design from fabrication, we empower researchers to design complex constructs irrespective of and independently from the manufacturing technique thus unlocking the full potential of synthetic biology. A typical example is plasmid assembly where the large number of standard plasmid architectures (e.g., SEVA, MoClo, GoldenGate, RFC10) and related assembly techniques actually hinders researchers’ design capabilities rather than enabling it. In this PhD project I focused on the development and validation of DNA fabrication workflows capable of handling multiple assembly techniques completely independent from the design input. The set of workflows developed allows researchers to submit their DNA design without any constraints related to fabrication techniques. The work presented here represents the foundational technology to enable a truly automated DNA foundry for synthetic biology.

The presence of RNA in the genome of living cells is one of the emerging topics of the last two decades and has been implicated in many biological processes. I focused my attention on ribonucleotides (rNMPs) embedded into DNA during genome duplication, as a threat to its integrity. In fact, rNMPs have been classified as the most frequent non-canonical nucleotides introduced during genome duplication by DNA polymerases. Such high incorporation frequency has been related to a physiological role in mismatch repair, but it can be easily turned into a source of genomic instability if rNMPs are not removed from DNA. This task is performed by RNase H activities that enable error-free repair of embedded single and multiple ribonucleotides. I first approached the issue of ribonucleotides incorporation into DNA from a physical point of view. Utilizing Atomic Force Microscopy I studied how ribonucleotides intrusions impact on DNA structure. The results obtained provided new insights on the structural changes imposed by ribonucleotides persistence into DNA. The other part of my Ph.D. project concerned the study of rNMPs incorporation in vivo, using the budding yeast S. cerevisiae as a model organism. The second aim was to unravel the function of the Translesion Synthesis polymerase η (Pol η) when the genome contains residual ribonucleotides and when deoxyribonucleotides (dNTPs) pools are depleted. We found that DNA polymerase η is responsible for the cell lethality observed when dNTPs are scarce and RNase H activities are defective. Therefore, I explored and characterized this unexpected toxic activity. We propose a model where Pol η supports cell survival in low dNTPs conditions by promoting DNA replication using ribonucleotides. While this activity is normally beneficial to wild type cells, it is highly toxic to cells defective for RNase H activities.

Theory of aging postulates that aging is a remodeling process where the body of survivors progressively adapts to internal and external damaging agents they are exposed to during several decades. Thus , stress response and adaptation mechanisms play a fundamental role in the aging process where the capability of adaptating effects, certainly, also is related the lifespan of each individual. A key gene linking aging to stress response is indeed p21, an induction of cyclin-dependent kinase inhibitor which triggers cell growth arrest associated with senescence and damage response and notably is involved in the up-regulation of multiple genes that have been associated with senescence or implicated in age-related . This PhD thesis project that has been performed in collaboration with the Roninson Lab at Ordway Research Institute in Albany, NY had two main aims: -the testing the hypothesis that p21 polymorphisms are involved in longevity -Evaluating age-associated differences in gene expression and transcriptional response to p21 and DNA damage In the first project, trough PCR-sequencing and Sequenom strategies, we we found out that there are about 30 polymorphic variants in the p21 gene. In addition, we found an haplotpype located in -5kb region of the p21 promoter whose frequency is ~ 2 fold higher in centenarians than in the general population (Large-scale analysis of haplotype frequencies is currently in progress). Functional studies I carried out on the promoter highilighted that the ―centenarian‖ haplotype doesn’t affect the basal p21 promoter activity or its response to p53. However, there are many other possible physiological conditions in which the centenarian allele of the p21 promoter may potentially show a different response (IL6, IFN,progesterone, vitamin E, Vitamin D etc). In the second part, project #2, trough Microarrays we seeked to evaluate the differences in gene expression between centenarians, elderly, young in dermal fibroblast cultures and their response to p21 and DNA damage. Microarray analysis of gene expression in dermal fibroblast cultures of individuals of different ages yielded a tentative "centenarian signature". A subset of genes that were up- or downregulated in centenarians showed the same response to ectopic expression of p21, yielding a putative "p21-centenarian" signature. Trough RQ-PCR (as well Microarrays studies whose analysis is in progress) we tested the DNA damage response of the p21-centenarian signature genes showing a correlation stress/aging in additional sets of young and old samples treated with p21-inducing drug doxorubicin thus finding for a subset of of them , a response to stress age-related.

Theory of aging postulates that aging is a remodeling process where the body of survivors progressively adapts to internal and external damaging agents they are exposed to during several decades. Thus , stress response and adaptation mechanisms play a fundamental role in the aging process where the capability of adaptating effects, certainly, also is related the lifespan of each individual. A key gene linking aging to stress response is indeed p21, an induction of cyclin-dependent kinase inhibitor which triggers cell growth arrest associated with senescence and damage response and notably is involved in the up-regulation of multiple genes that have been associated with senescence or implicated in age-related . This PhD thesis project that has been performed in collaboration with the Roninson Lab at Ordway Research Institute in Albany, NY had two main aims: -the testing the hypothesis that p21 polymorphisms are involved in longevity -Evaluating age-associated differences in gene expression and transcriptional response to p21 and DNA damage In the first project, trough PCR-sequencing and Sequenom strategies, we we found out that there are about 30 polymorphic variants in the p21 gene. In addition, we found an haplotpype located in -5kb region of the p21 promoter whose frequency is ~ 2 fold higher in centenarians than in the general population (Large-scale analysis of haplotype frequencies is currently in progress). Functional studies I carried out on the promoter highilighted that the ―centenarian‖ haplotype doesn’t affect the basal p21 promoter activity or its response to p53. However, there are many other possible physiological conditions in which the centenarian allele of the p21 promoter may potentially show a different response (IL6, IFN,progesterone, vitamin E, Vitamin D etc). In the second part, project #2, trough Microarrays we seeked to evaluate the differences in gene expression between centenarians, elderly, young in dermal fibroblast cultures and their response to p21 and DNA damage. Microarray analysis of gene expression in dermal fibroblast cultures of individuals of different ages yielded a tentative "centenarian signature". A subset of genes that were up- or downregulated in centenarians showed the same response to ectopic expression of p21, yielding a putative "p21-centenarian" signature. Trough RQ-PCR (as well Microarrays studies whose analysis is in progress) we tested the DNA damage response of the p21-centenarian signature genes showing a correlation stress/aging in additional sets of young and old samples treated with p21-inducing drug doxorubicin thus finding for a subset of of them , a response to stress age-related.

G-Quadruplexes (G4s) and R-loops are non-B DNA structures that can regulate transcription and replication. G4s are formed from four guanine residues that are held together in the same plane by Hoogsteen hydrogen bonds and further stabilized by the presence of monovalent cations. R-loops are triple-strand structures that contain an RNA-DNA hybrid and displaced single-stranded DNA. One of the most important features that influence these DNA structures is the GC content. Indeed, R-loop structures can be favoured by a high guanine density in the non-template DNA strand and this is specifically due to the higher thermodynamic stability of RNA-DNA hybrid. R-loops and G4s are generally regarded as highly deleterious, indeed the structures can block both transcription and DNA replication, creating replicative stress and potentially causing DNA damage. Here, we used immunofluorescence analysis in order to identify the increase of G4s and R-loops in cancer cells treated with specific G4 binders at long and very short time. At long time, the increase of these two non-B DNA structures triggers genomic DNA damage as established by the formation of γH2AX foci and other markers of cellular DNA damage response. Interestingly, stable and transient overexpression of RNaseH, an enzyme that specifically removes R-loop structures, induce a rescue of G4 binder-induced DNA damage and genome instability. Our study provides the first direct evidence of a mechanistic link between G4s and R-loops in human cancer cells.

Topoisomerase I (Top1) poisons are among the most clinically-effective drugs used for colon, ovary and lung cancers. Unpublished data from our lab have recently revealed that the structurally-unrelated Top1 poisons, Camptothecin (CPT) and Indimitecan (LMP776), induce the formation of micronuclei (MNi) in human cancer cells. In addition, MNi trigger an innate immune gene response by stimulating the cGAS/STING pathway. As the mechanisms of MNi formation are not fully determined, our aim is here to establish how MNi form after Top1 poisoning. Using immunofluorescence assays and EdU labelling of nascent DNAs, our results show that, after 24 hours of recovery, a short treatment with sub-cytotoxic doses of Top1 poisons induces the formation of MNi that do not contain newly synthetized (EdU+) DNA. We also saw that Top1 poisons delay replication machinery reducing EdU incorporation and produce significant levels of the damage markers γH2AX and p53BP1 in S-phase cells but not in G1 and G2/M cells. The results also show that MNi formation is dependent on R-loops, as RNaseH1 overexpression markedly reduces Top1 induced MNi. Genome-wide mapping of R-loops by DRIP-seq technique revealed that R-loop levels are both decreased and increased by CPT. In particular, increased R-loops are mainly found at active genes and always overlapped with Top1cc sites. We also found that increased R-loops overlap with lamina-associated chromatin domains while decreased R-loops correlate with replication origin sites. Overall, our data are consistent with the formation of MNi due to R-loop increase and under-replication at specific regions caused by Top1 poisons. These results will eventually help in developing new strategies for effective personalized interventions by using Top1-targeted compounds as immuno-modulators in cancer patients.

The DNA topology is an important modifier of DNA functions. Torsional stress is generated when right handed DNA is either over- or underwound, producing structural deformations which drive or are driven by processes such as replication, transcription, recombination and repair. DNA topoisomerases are molecular machines that regulate the topological state of the DNA in the cell. These enzymes accomplish this task by either passing one strand of the DNA through a break in the opposing strand or by passing a region of the duplex from the same or a different molecule through a double-stranded cut generated in the DNA. Because of their ability to cut one or two strands of DNA they are also target for some of the most successful anticancer drugs used in standard combination therapies of human cancers. An effective anticancer drug is Camptothecin (CPT) that specifically targets DNA topoisomerase 1 (TOP 1). The research project of the present thesis has been focused on the role of human TOP 1 during transcription and on the transcriptional consequences associated with TOP 1 inhibition by CPT in human cell lines. Previous findings demonstrate that TOP 1 inhibition by CPT perturbs RNA polymerase (RNAP II) density at promoters and along transcribed genes suggesting an involvement of TOP 1 in RNAP II promoter proximal pausing site. Within the transcription cycle, promoter pausing is a fundamental step the importance of which has been well established as a means of coupling elongation to RNA maturation. By measuring nascent RNA transcripts bound to chromatin, we demonstrated that TOP 1 inhibition by CPT can enhance RNAP II escape from promoter proximal pausing site of the human Hypoxia Inducible Factor 1 (HIF-1) and c-MYC genes in a dose dependent manner. This effect is dependent from Cdk7/Cdk9 activities since it can be reversed by the kinases inhibitor DRB. Since CPT affects RNAP II by promoting the hyperphosphorylation of its Rpb1 subunit the findings suggest that TOP 1inhibition by CPT may increase the activity of Cdks which in turn phosphorylate the Rpb1 subunit of RNAP II enhancing its escape from pausing. Interestingly, the transcriptional consequences of CPT induced topological stress are wider than expected. CPT increased co-transcriptional splicing of exon1 and 2 and markedly affected alternative splicing at exon 11. Surprisingly despite its well-established transcription inhibitory activity, CPT can trigger the production of a novel long RNA (5’aHIF-1) antisense to the human HIF-1 mRNA and a known antisense RNA at the 3’ end of the gene, while decreasing mRNA levels. The effects require TOP 1 and are independent from CPT induced DNA damage. Thus, when the supercoiling imbalance promoted by CPT occurs at promoter, it may trigger deregulation of the RNAP II pausing, increased chromatin accessibility and activation/derepression of antisense transcripts in a Cdks dependent manner. A changed balance of antisense transcripts and mRNAs may regulate the activity of HIF-1 and contribute to the control of tumor progression After focusing our TOP 1 investigations at a single gene level, we have extended the study to the whole genome by developing the “Topo-Seq” approach which generates a map of genome-wide distribution of sites of TOP 1 activity sites in human cells. The preliminary data revealed that TOP 1 preferentially localizes at intragenic regions and in particular at 5’ and 3’ ends of genes. Surprisingly upon TOP 1 downregulation, which impairs protein expression by 80%, TOP 1 molecules are mostly localized around 3’ ends of genes, thus suggesting that its activity is essential at these regions and can be compensate at 5’ ends. The developed procedure is a pioneer tool for the detection of TOP 1 cleavage sites across the genome and can open the way to further investigations of the enzyme roles in different nuclear processes.

The DNA topology is an important modifier of DNA functions. Torsional stress is generated when right handed DNA is either over- or underwound, producing structural deformations which drive or are driven by processes such as replication, transcription, recombination and repair. DNA topoisomerases are molecular machines that regulate the topological state of the DNA in the cell. These enzymes accomplish this task by either passing one strand of the DNA through a break in the opposing strand or by passing a region of the duplex from the same or a different molecule through a double-stranded cut generated in the DNA. Because of their ability to cut one or two strands of DNA they are also target for some of the most successful anticancer drugs used in standard combination therapies of human cancers. An effective anticancer drug is Camptothecin (CPT) that specifically targets DNA topoisomerase 1 (TOP 1). The research project of the present thesis has been focused on the role of human TOP 1 during transcription and on the transcriptional consequences associated with TOP 1 inhibition by CPT in human cell lines. Previous findings demonstrate that TOP 1 inhibition by CPT perturbs RNA polymerase (RNAP II) density at promoters and along transcribed genes suggesting an involvement of TOP 1 in RNAP II promoter proximal pausing site. Within the transcription cycle, promoter pausing is a fundamental step the importance of which has been well established as a means of coupling elongation to RNA maturation. By measuring nascent RNA transcripts bound to chromatin, we demonstrated that TOP 1 inhibition by CPT can enhance RNAP II escape from promoter proximal pausing site of the human Hypoxia Inducible Factor 1 (HIF-1) and c-MYC genes in a dose dependent manner. This effect is dependent from Cdk7/Cdk9 activities since it can be reversed by the kinases inhibitor DRB. Since CPT affects RNAP II by promoting the hyperphosphorylation of its Rpb1 subunit the findings suggest that TOP 1inhibition by CPT may increase the activity of Cdks which in turn phosphorylate the Rpb1 subunit of RNAP II enhancing its escape from pausing. Interestingly, the transcriptional consequences of CPT induced topological stress are wider than expected. CPT increased co-transcriptional splicing of exon1 and 2 and markedly affected alternative splicing at exon 11. Surprisingly despite its well-established transcription inhibitory activity, CPT can trigger the production of a novel long RNA (5’aHIF-1) antisense to the human HIF-1 mRNA and a known antisense RNA at the 3’ end of the gene, while decreasing mRNA levels. The effects require TOP 1 and are independent from CPT induced DNA damage. Thus, when the supercoiling imbalance promoted by CPT occurs at promoter, it may trigger deregulation of the RNAP II pausing, increased chromatin accessibility and activation/derepression of antisense transcripts in a Cdks dependent manner. A changed balance of antisense transcripts and mRNAs may regulate the activity of HIF-1 and contribute to the control of tumor progression After focusing our TOP 1 investigations at a single gene level, we have extended the study to the whole genome by developing the “Topo-Seq” approach which generates a map of genome-wide distribution of sites of TOP 1 activity sites in human cells. The preliminary data revealed that TOP 1 preferentially localizes at intragenic regions and in particular at 5’ and 3’ ends of genes. Surprisingly upon TOP 1 downregulation, which impairs protein expression by 80%, TOP 1 molecules are mostly localized around 3’ ends of genes, thus suggesting that its activity is essential at these regions and can be compensate at 5’ ends. The developed procedure is a pioneer tool for the detection of TOP 1 cleavage sites across the genome and can open the way to further investigations of the enzyme roles in different nuclear processes.

Recenti analisi sull’intero trascrittoma hanno rivelato una estensiva trascrizione di RNA non codificanti (ncRNA), le quali funzioni sono tuttavia in gran parte sconosciute. In questo lavoro è stato dimostrato che alte dosi di camptotecina (CPT), un farmaco antitumorale inibitore della Top1, aumentano la trascrizione di due ncRNA antisenso in 5’ e 3’ (5'aHIF-1α e 3'aHIF-1α rispettivamente) al locus genico di HIF-1α e diminuiscono i livelli dell’mRNA di HIF-1α stesso. Gli effetti del trattamento sono Top1-dipendenti, mentre non dipendono dal danno al DNA alla forca di replicazione o dai checkpoint attivati dal danno al DNA. I ncRNA vengono attivati in risposta a diversi tipi di stress, il 5'aHIF-1α è lungo circa 10 kb e possiede sia il CAP in 5’ sia poliadenilazione in 3’ (in letteratura è noto che il 3'aHIF-1α è un trascritto di 1,7 kb, senza 5’CAP né poliadenilazione). Analisi di localizzazione intracellulare hanno dimostrato che entrambi sono trascritti nucleari. In particolare 5'aHIF-1α co-localizza con proteine del complesso del poro nucleare, suggerendo un suo possibile ruolo come mediatore degli scambi della membrana nucleare. È stata dimostrata inoltre la trascrizione dei due ncRNA in tessuti di tumore umano del rene, evidenziandone possibili ruoli nello sviluppo del cancro. È anche noto in letteratura che basse dosi di CPT in condizioni di ipossia diminuiscono i livelli di proteina di HIF-1α. Dopo aver dimostrato su diverse linee cellulari che i due ncRNA sopracitati non potessero essere implicati in tale effetto, abbiamo studiato le variazioni dell’intero miRnoma alle nuove condizioni sperimentali. In tal modo abbiamo scoperto che il miR-X sembra essere il mediatore molecolare dell’abbattimento di HIF-1α dopo trattamento con basse dosi di CPT in ipossia. Complessivamente, questi risultati suggeriscono che il fattore di trascrizione HIF-1α venga finemente regolato da RNA non-codificanti indotti da danno al DNA.
Whole transcriptome analyses revealed a broad transcription of non-coding RNAs (ncRNA), which functions are largely unknown. In this work it was shown that high doses of camptothecin (CPT), an antitumor inhibitor of Top1, increase the transcription of two ncRNA antisense 5 'and 3' (5'aHIF-1α and 3'aHIF-1α respectively) respect to the locus HIF-1α gene and decreased HIF-1α mRNA levels. Treatment effects are Top1-dependent, while are not dependent by the replication fork-mediated DNA damage or by DNA damage-activated checkpoints. The ncRNAs are activated in response to different stress types, the 5'aHIF-1α is about 10 kb length and it has both 5’CAP and polyadenylation (in literature it is known that the 3'aHIF-1α is a transcript of 1.7 kb, with no 5'CAP and no polyadenylation). Intracellular localization have shown that both ncRNAs are nuclear transcripts. In particular 5'aHIF-1α co-localizes with nuclear pore complex proteins, suggesting its possible role as a traffic-mediator of the nuclear membrane. It has been demonstrated also the transcription of the two ncRNAs in human kidney tumor tissues, highlighting it possible roles in cancer development. It is also known that low doses of CPT in hypoxia conditions decrease the HIF-1α protein levels. Having demonstrated on several cell lines that the two ncRNA above could not be implicated in this effect, we studied the entire human miRnoma variations under our experimental conditions. Thus we found that miR-X seems to be the molecular mediator of HIF-1α abatement after low doses CPT treatment in hypoxia conditions. Overall, these results suggest that HIF-1α transcription factor is finely regulated by non-coding RNA induced by DNA damage.

Recenti analisi sull’intero trascrittoma hanno rivelato una estensiva trascrizione di RNA non codificanti (ncRNA), le quali funzioni sono tuttavia in gran parte sconosciute. In questo lavoro è stato dimostrato che alte dosi di camptotecina (CPT), un farmaco antitumorale inibitore della Top1, aumentano la trascrizione di due ncRNA antisenso in 5’ e 3’ (5'aHIF-1α e 3'aHIF-1α rispettivamente) al locus genico di HIF-1α e diminuiscono i livelli dell’mRNA di HIF-1α stesso. Gli effetti del trattamento sono Top1-dipendenti, mentre non dipendono dal danno al DNA alla forca di replicazione o dai checkpoint attivati dal danno al DNA. I ncRNA vengono attivati in risposta a diversi tipi di stress, il 5'aHIF-1α è lungo circa 10 kb e possiede sia il CAP in 5’ sia poliadenilazione in 3’ (in letteratura è noto che il 3'aHIF-1α è un trascritto di 1,7 kb, senza 5’CAP né poliadenilazione). Analisi di localizzazione intracellulare hanno dimostrato che entrambi sono trascritti nucleari. In particolare 5'aHIF-1α co-localizza con proteine del complesso del poro nucleare, suggerendo un suo possibile ruolo come mediatore degli scambi della membrana nucleare. È stata dimostrata inoltre la trascrizione dei due ncRNA in tessuti di tumore umano del rene, evidenziandone possibili ruoli nello sviluppo del cancro. È anche noto in letteratura che basse dosi di CPT in condizioni di ipossia diminuiscono i livelli di proteina di HIF-1α. Dopo aver dimostrato su diverse linee cellulari che i due ncRNA sopracitati non potessero essere implicati in tale effetto, abbiamo studiato le variazioni dell’intero miRnoma alle nuove condizioni sperimentali. In tal modo abbiamo scoperto che il miR-X sembra essere il mediatore molecolare dell’abbattimento di HIF-1α dopo trattamento con basse dosi di CPT in ipossia. Complessivamente, questi risultati suggeriscono che il fattore di trascrizione HIF-1α venga finemente regolato da RNA non-codificanti indotti da danno al DNA.
Whole transcriptome analyses revealed a broad transcription of non-coding RNAs (ncRNA), which functions are largely unknown. In this work it was shown that high doses of camptothecin (CPT), an antitumor inhibitor of Top1, increase the transcription of two ncRNA antisense 5 'and 3' (5'aHIF-1α and 3'aHIF-1α respectively) respect to the locus HIF-1α gene and decreased HIF-1α mRNA levels. Treatment effects are Top1-dependent, while are not dependent by the replication fork-mediated DNA damage or by DNA damage-activated checkpoints. The ncRNAs are activated in response to different stress types, the 5'aHIF-1α is about 10 kb length and it has both 5’CAP and polyadenylation (in literature it is known that the 3'aHIF-1α is a transcript of 1.7 kb, with no 5'CAP and no polyadenylation). Intracellular localization have shown that both ncRNAs are nuclear transcripts. In particular 5'aHIF-1α co-localizes with nuclear pore complex proteins, suggesting its possible role as a traffic-mediator of the nuclear membrane. It has been demonstrated also the transcription of the two ncRNAs in human kidney tumor tissues, highlighting it possible roles in cancer development. It is also known that low doses of CPT in hypoxia conditions decrease the HIF-1α protein levels. Having demonstrated on several cell lines that the two ncRNA above could not be implicated in this effect, we studied the entire human miRnoma variations under our experimental conditions. Thus we found that miR-X seems to be the molecular mediator of HIF-1α abatement after low doses CPT treatment in hypoxia conditions. Overall, these results suggest that HIF-1α transcription factor is finely regulated by non-coding RNA induced by DNA damage.

The MRN complex and the cellular response to DNA double-strand breaks The Mre11/Rad50/Nbs1 (MRN) complex is a multisubunit nuclease that plays a crucial role in DNA double-strand break (DSB) repair and ATM (Ataxia-Telangiectasia mutated)-mediated checkpoint signaling. The first part of this thesis addresses the controversy over MRN complex involvement in the non-homologous end-joining (NHEJ) DSB repair pathway by the use of a plasmid DSB repair assay in Xenopus laevis cell-free extracts. Mre11-depleted extracts are able to support efficient NHEJ repair of DSBs, regardless of the end-structure. Mre11-depletion does not alter the kinetics of end-joining or the type and frequency of junctions found in repaired products. Finally, Ku70-independent end-joining events are not affected by Mre11 loss. Our data demonstrate that the MRN complex is not required for efficient and accurate NHEJ-mediated repair of DSBs in this vertebrate system. In the second part, I discuss the techniques, molecular tools and preliminary results of the characterization of the molecular mechanisms underlying the involvement of the MRN-ATM pathway in the maintenance of genome integrity during DNA replication.

Congenital hypothyroidism (CH) is the most frequent endocrinopathy in newborn. If not promptly treated lead to a severe impairment of psychomotor development. The etiophatogenesis of CH is still poorly understood; although several causative genes are identified, they can explain only a small portion of the pathological phenotypes. This scenario is further complicated if we focused on the incidence of CH in different context. In fact, epidemiological data indicate that children born prematurely have a 3-5 fold higher risk of CH. In addition premature infants born small for gestational age (SGA) have a risk of 12% higher to develop IC compared to prematures with appropriate development (AGA). The mechanisms that justify the increased risk of IC are still unknown. Some studies report that aberrant methylation patterns are associated with prematurity, intrauterine fetal development and the onset of some diseases. This project is focused on the study of DNA methylation, as predisposing factor to permanent thyroid dysfunction with neonatal onset. Using the Illumina Infinium-HumanMethylation27 technology we analyzed the global DNA methylation patterns (AVG) and selected the differentially methylated genes (DMGs) between 31 CH-cases born premature, AGA or SGA, and 28 term or preterm controls. To better understand the relationship between the DNA methylation and the premature birth, the intrauterine growth and the thyroid defect, the following groups were selected according to the gestational age at birth: 12 CH-with very preterm birth (CH-VPB<32wks) and 19 CH-with preterm birth (CH-PB 32-37wks); Controls: 9-CVPB, 6-CPB, 12-term birth (CTB>37wks). The same subjects were then analyzed according to intrauterine growth (20 CH-SGA, 11 CH-AGA than 6 C-SGA and 20 C-AGA) or the degree of CH: 19 with overt CH (OH, TSH>10 U/L) and 12 with mild CH (MH, TSH<10 U/L) than 16 CPB and 12 CTB. The global methylation analysis showed that infants born prematurely and SGA have a significant hypomethylation than term-controls. These data were confirmed by the gene-specific methylation analysis, through which we selected a large group of differentially methylated genes (DMGs) in CH-cases than term controls. Interestingly, the 95% of the DMGs are hypomethylated and the 70% of them are represented by CpG sites located in DNA non-coding regions. The gene ontology analysis revealed that genes involved in fetal growth and thyroid hormone metabolism were included among DMGs. The analysis of nine maternal genomic DNA for polymorphisms at the MTHFR revealed the possible association with folate deficiency during pregnancy and the global hypomethylation status of affected newborns. This is the first work exploring the role of epigenetic influences in the predisposition to congenital hypothyroidism. Our results suggest that genomic instability caused by global hypomethylation of non-coding regions may be related to premature birth and fetal growth delay. Under these conditions, thyroid defects are more frequent than expected and could result from the increased expression of predisposing genes, rather than from the reduced expression of protective genes. The role of maternal conditions during pregnancy seems to be a key factor to determine a proper DNA methylation pattern on fetus. Based on the Developmental Origin of Health and Disease Theory, we can assume that adverse condition during pregnancy, such as folate deficiency, may produce a fetal epigenetic reprogramming and the adaptation of preterm neonate to the extrauterine life includes among other dysfunctions a thyroid functional impairment. If this data will be confirmed by further experiments, this could represent a new predisposing factor to take into account during pregnancy to prevent and improve the prenatal screening of CH.

The cyclin-dependent kinase inhibitor p21CDK1NA is a protein involved in various cellular processes, such as cell cycle arrest, transcription regulation, apoptosis and cell motility. p21 was discovered to actively participate also to DNA repair process, such as nucleotide excision repair (NER), through the interaction with Proliferative Cell Nuclear Antigen (PCNA). For this study, we used plasmids driving the expression of three different mutated form of p21 with a fluorescent tag (GFP) fused to their C-term: p21DD-GFP (T148D), p21AAA-GFP (KRR154-156AAA) and p212KQ-GFP (K161,163Q). These mutants, reported in literature to be resistant to degradation, have been used to test whether the PCNA-interacting partners dynamics at the DNA damage sites (e.g., the recruitment and subsequent release from the repair site) is influenced by the p21 susceptibility to degradation, while the interaction with PCNA is conserved. We initially performed fluorescence microscopy analysis and immunoprecipitation assays on transfected HeLa cells to characterize the p21-GFP mutants. The results showed that, although both p21DD-GFP and p212KQ-GFP mutants have a clear nuclear localization, unexpectedly p21AAA-GFP did not interact with PCNA inhibiting its recruitment to DNA damage sites. Subsequently, we confirmed the enhanced stability of p21AAA and p212KQ mutants, while p21DD showed a poor resistance to UVC-induced degradation. To test whether p21 could influence the PCNA-interacting partners turnover, we compared the persistence of p212KQ with p21WT at the DNA damage sites performing an immunofluorescence assay on HeLa cells transfected with HA-p21WT and HA-p212KQ. The results showed a longer retention of DNA repair factors (e.g., DNA polymerase δ) at the DNA lesions in the presence of p21 mutant, suggesting a consequent delay in the DNA repair process. Then, we also performed live cell imaging confocal analyses using HeLa cell line stably expressing mCherry-PCNA co-transfected with two plasmids driving the expression of fluorescent-tagged (miRFP) DNA Ligase 1 and either p21WT-GFP or p212KQ-GFP, respectively. PCNA and DNA Lig 1 release kinetics in the absence p21 and in the presence of p212KQ resulted to be remarkably slower as compared to p21WT. Therefore, the data suggest that not only the persistence of p21 at the DNA damage sites, but even the absence of p21 could deregulate the DNA repair process. Along with these results, a significant reduction of NER efficiency was detected in HeLa cells transfected with p212KQ-GFP plasmid and in the absence of p21. Consistently, we observed a similar reduction in DNA re-synthesis after UV-C irradiation in human fibroblasts lacking p21. In conclusion, these results suggest a possible mechanism through which p21 influences the PCNA partner dynamics by coupling to the degradation process, which is fundamental to finely tune p21 cellular levels.

A ROLE OF Caenorhabditis elegans polq-1 AND hel-308 IN DNA REPAIR By Dott. Diego Matteo Muzzini Genomes are daily challenged by different mutagens, which can seriously damage it. Since un-repaired DNA damage leads to apoptosis or cancer development, genomes evolved a number of different DNA repair pathways to face such damages. Among all possible DNA lesions, Interstrand Cross-Links (ICLs), which covalently bind the DNA strands together, are the most toxic ones. A number of evidences demonstrated that ICL could be repaired using Homologous Recombination (HR), Fanconi Anemia (FA) and Tranlesion Synthesis (TLS) pathways alone or in combination. Nevertheless, a precise mechanistic model is still lacking in eukaryotes and it is thought that many genes could be involved to overcome this kind of lesion. The Drosophila melanogaster gene mus-308 and spnC/mus301 are genes involved in DNA damage repair. I particulat they seems dedicated to ICLs lesions repair. In my PhD project I focused my effort to elucidate the role of polq-1 and hel-308, which are Caenorhabditis elegans orthologs of mus-308 and spnC/mus301, during ICL repair. Here I show that polq-1 and hel-308 are the functional homologues of mus-308 and spnC/mus301, respectively and they are necessary for DNA repair in worms. Moreover, polq-1 seems not to involved neither in homologous recombination (HR) nor in the Fanconi anemia (FA) pathways, while it synergically interacts with hel-308 in a non-linear pathway, probably also involving translesion DNA synthesis (TLS).

DNA damage response (DDR) mechanisms are crucial in all organisms to detect DNA damage and integrate its repair with cell cycle control, chromatin structure, and other adaptive changes in cell physiology. The highly conserved structural maintenance of chromosomes (SMC) complex, SMC5/6, contains both SUMO and ubiquitin ligase activities in its composition and is crucial for processing recombination structures arising during replication. Differently from related SMC complexes such as Cohesin and Condensin, the function of SMC5/6 in genome maintenance and its mode of action remain to date hardly understood. Moreover, a lack of cellular models for SMC5/6, especially in human cell lines, has hampered progress in understanding its roles in DDR in model systems most relevant for human disease. Here, we established vertebrate and human cellular models of SMC5/6 knockouts and characterized the associated phenotypes in a battery of assays. Specifically, I established (conditional) SMC5 knockout (KO) in chicken (DT40) and human (TK6) B cell lines. We generated a conditional SMC5 KO in TK6 human cell lines, and uncovered that SMC5 depletion caused apoptosis-mediated lethality, associated with increased DSBs and cohesion defects. In chicken DT40 cell lines, SMC5 KO causes a slow growth phenotype depending on the temperature condition. Second, we investigated the roles of SMC5/6 in intra- and inter-strand crosslink (ICL) repair. SMC5 shows epistasis with FA core mutations, as well as with DDX11 and RAD17, without affecting FANCD2-I ubiquitination and CHK1 phosphorylation. We propose that SMC5 is working downstream of FA and RAD17/DDX11-mediated pathways, likely in resolving the emerging recombination intermediates. Third, we are attempting to identify the principles underlying SMC5/6 roles in the establishment/maintenance of chromatin structure/cohesion and integrity of specific genomic loci such as replicating fragile sites and centromeric/peri-centromeric regions. To these ends, we have used BLISS, a genomic approach to map DSBs, and plan to employ ChIP-seq of endogenously tagged SMC5/6.

Checkpoints are surveillance mechanisms that monitor cell cycle progression and preserve the correct order of events. Checkpoints are also activated in response to genomic insults, or alterations of cellular structures, and lead to temporary cell cycle arrest, slowing down of DNA replication, changes in the cellular transcriptional program and, in some instances, apoptosis. The mechanisms used by checkpoints to identify DNA lesions are poorly understood and may involve the function of repair proteins. Looking for mutants specifically defective in activating the checkpoint following UV lesions, but proficient in the response to methyl methane sulfonate and double-strand breaks, we isolated RAD14, the homolog of human XPA, involved in lesion recognition during nucleotide excision repair (NER). Rad14 was also isolated as a partner of the Ddc1 checkpoint protein in a two-hybrid screening, and physical interaction was proven by co- immunoprecipitation. We show that lesion recognition is not sufficient for checkpoint activation, but processing, carried out by repair factors, is required for recruiting checkpoint proteins to damaged DNA. Mutations affecting the core NER machinery abolish G1 and G2 checkpoint responses to UV, preventing activation of the Mec1 kinase and its binding to chromosomes. Conversely, elimination of transcription coupled or global genome repair alone does not affect checkpoints, suggesting a possible interpretation for the heterogeneity in cancer susceptibility observed in different NER syndrome patients. Moreover we show that in Saccharomyces cerevisiae epigenetic modification of histones is required for checkpoint activity in response to a variety of genotoxic stresses. We demonstrate that ubiquitination of histone H2B on lysine 123 by the Rad6-Bre1 complex, is necessary for activation of Rad53 kinase and cell cycle arrest. We found a similar requirement for Dot1-dependent methylation of histone H3. Loss

Ddc1p is a member of the DNA damage checkpoint, a conserved surveillance mechanism whose main function is to delay cell cycle progression in the presence of damaged chromosomes, allowing time for DNA repair. Ddc1p is phosphorylated periodically during a normal cell cycle and becomes hyperphosphorylated in response to DNA damage, but nothing is currently known about the functional meaning of this modification. By mutating Ddc1 s putative phosphorylation sites, we have identified a role for the post-traductional modification in checkpoint inactivation in response to DNA double strand brakes: the mutant strain, in fact, is able to adapt to irreparable DSB earlier than the wild type, and this effect is increased when the protein is overexpressed. Moreover, even the overexpression of wild type Ddc1p induces a faster adaptation; a possible explanation is that Ddc1p overexpression regulates the rate of DNA resection at the lesion, which is strictly correlated with the processes of checkpoint activation and inactivation.

Questa tesi tratta delle k-forme differenziali come utili strumenti in ambito fisico e biochimico. Dopo averle definite, si osserveranno alcune proprietà e operatori importanti, quali il pullback, la derivazione esterna e l'operatore * di Hodge. Sulla base di queste conoscenze verranno riscritte le equazioni di Maxwell in forma più compatta attraverso le k-forme. In seguito verrà introdotto il concetto di "linking number" mediante la definizione di grado di una funzione. Grazie alla teoria dei nodi sarà possibile studiare questo invariante topologico nel contesto del DNA e delle proteine: non esiste un metodo sperimentale per osservare direttamente la dinamica dell'azione enzimatica, ma grazie agli studi sui cambiamenti nella geometria del DNA e delle proteine è ora possibile dedurre i meccanismi degli enzimi e prevenire alcune patologie dovute a folding errati.

Holliday junction-resolving enzymes (X-resolvases) mediate the reaction by recognizing DNA four-way junctions and introducing symmetrical nicks in a very precise manner. They are dimeric proteins that recognize DNA four-way junctions and resolve them with high accuracy by introducing symmetrical nicks on both sides of the branch point. X-resolvases exhibit a stretch of basic amino acids on their surface that are responsible for DNA binding. I have set out to study the structure of the archaeal resolvase Holliday junction resolvase from Archaeoglobus fulgidus, alone and in complex with 4-way junction, using X-ray crystallography. The crystal structure of Hjc alone was solved at 1.63 Å resolution, and it shows the same overall fold of other Hjc family members. The structure was not complete, due to the distortion of some parts. In order to obtain the complete structure, a surface mutant was created by site mutagenesis of two non-conserved residues. The surface mutant structure, solved at 1.56 Å, shows a complete protein, and the catalytic flexible loop can be observed distorted in two different functional conformations.I also report the crystal structure of Hjc in complex with the 4-way junction with 10bp long arms, at 3.15 Å resolution. This structure provides the first picture of a Holliday junction bound to the Holliday junction cutting enzyme in archaea. The structural model shows a novel form of protein- Holliday junction binding, the biological meaning of which is yet to be clarified.

Genomic instability is commonly associated with pathological disorders including cancer. The progressive accumulation of genetic abnormalities in cancer-associated genes can confer cellular autonomous proliferation, contributing to the oncogenic transformation. Although animal models have been instrumental to the understanding of the molecular mechanisms responsible for tumor progression, there are severe limitations for success. This thesis work aimed at the development of an innovative animal model that could recapitulate the ongoing lifelong accumulation of DNA lesions, leading to neoplastic transformation. To this end, the natural DNA mutating enzyme AID was fused to sequence specific DNA binding proteins. They were engineered to target tumor suppressor genes and to induce low-frequency mutagenesis in cell lines and in zebrafish. A TALE-Aid fusion protein was targeted to the p53 locus of mouse cell lines, and the Aid-dependent mutations were monitored by next-generation sequencing. The induced mutations occurred at a comparable frequency to those observed in Aid-induced non-immunoglobulin gene targets in B cells. Mutations were found mostly in the DNA binding domain of p53, possibly reflecting AID- hotspot residues in p53. Our approach also induced mutations that have not been characterized previously, and could provide further insight into p53 dependent oncogenesis. When TALE-AID were expressed and targeted to the p53 locus of zebrafish embryos, the activity induced developmental abnormalities with variable severity, leading to both an increased mortality, and impaired ovarian maturation and fertility. We have developed and initially characterized a novel tool for the in vitro / in vivo study of the accumulation of mutations in cancer-associated genes.

In pre-cancerous lesions the overexpression of oncogenes such as Myc not only drives aberrant cellular proliferation, but also triggers a strong DNA damage response (DDR) that is in part due to DNA damage accumulating at the level of stalled replication forks. This oncogene-induced DDR is an effective barrier to cancer development and represents a relevant tumor suppressive mechanism. Conversely, at later stages of malignancy DDR signaling may function in favor of cancer progression. Such tumor promoting role of DDR may be needed for cancer cells to avoid accumulation of cytotoxic DNA damage under high level of oncogene-induced replication stress. Recently it has been shown that targeting regulators of replication checkpoint such as ATR or Chk1 in Myc-overexpressing cells caused apoptosis and prevented tumor formation, suggesting a crucial role for this pathway in ensuring cancer cell viability and offering the chance of developing new targeted therapies against cancer cells. In an effort to identify the modulators of Myc-induced replicative stress, we carried out a high-throughput RNAi screen based on immunofluorescence detection of ϒH2AX, a DNA damage marker. Quantification of the number of cells and the percentage of ϒH2AX-positive cells, identified hits that exhibited differential cell viability (synthetic lethal hits) and/or enhanced ϒH2AX signal (DDR-up hits) in Myc-overexpressing cells compared to normal cells. Validated hits encompass a variety of pathways and biological processes and have different molecular functions. As a proof of principal, we selected SRSF3 and Cdk12 and confirmed the synergistic effect of Myc overexpression and depletion of SRSF3 or Cdk12 on accumulation of cytotoxic DNA damage response as marked by H2AX phosphorylation. For further mechanistic investigations, we selected Rad21, a component of the cohesin complex, which was also reported as a Myc-synthetic lethal candidate previously. Using small inhibitory RNAs against Rad21, we confirmed that depletion of Rad21, increased ϒH2AX level and subsequently led to cell death, selectively in Myc-overexpressing cells. We provide evidence that while Rad21 is necessary for proper and efficient DNA synthesis, replication reinforcement imposed by Myc overexpression in Rad21-depleted cells results in replicative stress. In addition, we observed that Myc, as a transcription factor, could partially rescue transcriptional alterations due to Rad21 depletion. The conflicts between DNA replication and transcription in Rad21-depleted cells upon Myc activation may be the source of increased R-loops detected in these cells. In summary, by means of a genetic loss of function screen we identified several candidates that may be involved in protecting Myc-overexpressing cells against ample replicative stress, thus revealing targets for potential therapeutic intervention in Myc-driven cancers.

DNA is the most precious molecule in our cells, thus it has to be protected from damage and alterations and, if damaged, it has to be repaired efficiently. The DNA damage response (DDR) is a signaling cascade that follows the generation of a lesion in the DNA double helix and promptly arrests cell proliferation in order to attempt DNA repair. It has been proposed that mammalian genomes are pervasively transcribed, also in non-coding regions. Non-coding RNAs (ncRNAs) have been involved in an increasing number of cellular events and some of them are processed by members of the RNA interference (RNAi) pathway. So far, RNAi and DDR pathways have not been demonstrated to directly interact. During my PhD, I contributed to uncover an unexpected layer of DDR regulation by a new class of DICER- and DROSHA-dependent small non-coding RNA, named DDRNA. DDR foci stability is sensitive to RNA polymerase II inhibition and to RNase A treatment. Incubation of RNase A-treated cells with DICER- and DROSHA-dependent RNA products restores focal accumulation of DDR factors. DICER and DROSHA are indeed necessary to trigger DDR upon exogenous DNA damage in human cells, and DICER processing activity is necessary to activate DDR. Moreover, DICER and DROSHA knockdown impacts on checkpoint activation and allows senescent cells to re-enter S-phase. Differently, inactivation of GW182, a component of the RNAi machinery involved downstream of DICER and DROSHA in mRNA translational control, does not impact on DDR foci formation and detection. In a mammalian cell system in which a single DNA double-strand break can be generated at a defined exogenous integrated locus, DDR focus formation requires site-specific RNA molecules. RNA deep sequencing confirmed the presence of 22-23-nucleotide sequence-specific transcripts arising from the damaged locus, which are DICER-dependent. These DDR-regulating RNAs (DDRNAs) act at the first steps of the DDR cascade, in an MRN-dependent manner and have an impact also on DNA damage repair. Importantly, DDRNAs, both chemically synthesized or generated in vitro by DICER cleavage, are biologically active and antisense LNA oligonucleotides reduce DDR activation in living cells. Finally, by the use of fluorescently labeled molecules, DDRNAs have been demonstrated to localize at the site-specific damaged locus. Collectively these results suggest an unanticipated direct role of DICER and DROSHA in the production of small ncRNAs that control DDR activation at sites of DNA damage. Given the known tumor suppressive functions of DDR and the implication of its activation in a number of biological relevant processes, such as senescence, this discovery may have a significant impact on our understanding of ageing and cancer.

Our group previously demonstrated that when a DNA double-strand break (DSB) occurs, RNA polymerase II (RNAPII) is recruited to the exposed DNA ends and allows the by-directional synthesis of transcripts derived from exposed DNA ends that we called damage-induced long non-coding RNAs (dilncRNAs). Recently we reported that a DSB also recruits the full transcriptional machinery, in particular the preinitiation complex (PIC), just as commonly recruited at canonical RNA polymerase II-driven transcriptional promoters. This, blurs the distinction between a DSB and a transcriptional promoter. dilncRNAs were defined as non-coding as most of our genome is not coding for polypeptides and thus, DSB will most often transcribe non coding regions of DNA. However, what would be the consequences of a DSB occurring upstream of a gene unit, lacking a promoter but carrying all other features of a coding sequence, such as an open reading frame (ORF) and a poly(A) signal? Would the ensuing dilncRNA actually be a coding transcript? In other words: can a DSB trigger the synthesis of a protein? Therefore, the aim of this project is to test if a DSB appropriately positioned at the transcriptional start site (TSS) of an otherwise silent gene may assemble a functional promoter, triggering RNA synthesis which leads to protein expression. I have carried out such studies in artificial cell systems in which I have engineered promoter-less reporter genes. In addition, I have tested whether a DSB is sufficient to re-express a candidate tumour suppressor gene, HIC-1 (hyper-methylated in cancer 1), that is commonly silenced by promoter methylation. In more in detail, I tested my working hypothesis in three different systems. As a proof of concept, I first used two reporter gene-based systems: a stable clonal cell line of HeLa cells bearing an integrated lentiviral construct encoding for an enhanced green fluorescent protein (EGFP) ORF lacking its transcriptional promoter and enhancer region, and an immortalized mouse embryonic fibroblast (MEF) cell line carrying a promoter-less enhanced yellow fluorescent protein (EYFP) integrated by homologous recombination in the locus Rosa26 of the mouse genome. To induce sites-specific DSBs, I employed the CRISPR/Cas9 technology. Single guide RNAs targeting the GFP or EYFP transcriptional start site (TSS) were cloned in a lentiviral vector and cells were transduced with such a vector expressing them together with Cas9 to test the impact of DSB induction on the expression of the reporter gene. The second system takes advantage of MDA MB 231, a human breast cancer cell line, in which HIC1 endogenous gene is actively silenced by promoter DNA methylation. The goal, here, is to infect these cells with a CRISPR/Cas9 lentiviral vector to induce a DSB at the TSS of HIC1 gene and monitor the synthesis of a mature messenger RNA. These experiments suggested that a DSB, appropriately positioned at a gene TSS, triggers the transcription of RNAs that are polyadenylated, exported in the cytoplasm and translated into a functional protein. This study could be relevant to identify a novel mechanism of transcriptional regulation based on DSB at promoters. This approach could also be used to activate gene expression in genetic disease settings, or to activate the expression of tumor suppressor genes that are silenced in tumors.

Human Exo1 is required to activate the checkpoint response after UV irradiation in human cells. hExo col-localizes with NER factors at the sites of UV lesions and hEXO1 down-regulation by siRNA technology affects DNA repair synthesis.

The DNA damage response is a pathway responsible for the maintenance of genome integrity. In my thesis I focused on the investigation of the modulation of ATM activity, a DNA damage response master kinase, by NOTCH1 receptor. Here I show that NOTCH1 inhibits DNA damage response activation. This inhibitory effect of NOTCH1 is not mediated by its transcriptional activity, but it is the result of direct binding between NOTCH1 and ATM kinase. I show that NOTCH1 binds to the FATC domain of ATM, and this results in an inhibition of ATM kinase activity. Furthermore, I provide evidence that NOTCH1-mediated ATM inhibition does not result from the impairment of ATM recruitment to DNA double-strand breaks. Rather, I show that NOTCH1 competes with FOXO3a transcription factor for the binding to the FATC domain of ATM and that over-expression of FOXO3a prevents NOTCH1-mediated ATM inhibition. As the exact function of FOXO3a in ATM activation was unclear, I sought to understand molecular mechanisms underlying NOTCH1-mediated ATM inactivation and the role of FOXO3a as an opposing factor in this process. I discovered that FOXO3a forms a direct complex with KAT5 lysine acetyl transferase that is critical for ATM activation upon DNA damage. Moreover, I observed that FOXO3a was necessary for the formation of a complex between ATM and KAT5. Surprisingly, I observed that NOTCH1 was not only impairing ATM-KAT5 interaction, but also FOXO3a-KAT5 one. This unexpected observation led me to the discovery that FOXO3a-KAT5 interaction is restricted to the formation of this three-protein complex together with the ATM kinase. Next, I demonstrated that induction of FOXO3a nuclear localization as well as inhibition of NOTCH1 increases ATM activation in NOTCH1-driven cancer cells, which leads to augmented DNA damage-induced cell death. Finally, I show that, in addition to ATM, NOTCH1 interacts also with other PI3K-like kinases: DNA-PKcs and ATR. Although I did not observe a significant impact of NOTCH1 on ATR kinase activation in the experimental settings I used, I observed an impaired activation of DNA-PKcs, which however did not result in a significant reduction of DNA damage repair in NOTCH1-expressing cells.

The c-Myc oncoprotein (or Myc) is a transcription factor of the basic-Helix-Loop-Helix Leucine-zipper (bHLH-LZ) family, whose transcriptional activity depends on dimerization with the bHLH-LZ partner Max and DNA binding, mediated by the basic regions of both proteins. Myc/Max dimers bind preferentially to the hexanucleotide motif CACGTG (known as E-box) and variants thereof. The ability of Myc to bind DNA in vivo, however, is not stringently regulated by the presence of the E-box, since many genomic sites targeted by Myc do not contain this motif. Hence, we still need to fully comprehend how Myc recognizes its genomic targets and to what extent sequence-specific DNA binding contributes to this process. Based on the crystal structure of the DNA-bound Myc/Max dimer, we generated a Myc mutant in which two basic region residues engaged in sequence-specific contacts (H359 and E363) were mutated to Alanine (Myc HEA), and compared this with a mutant in which three Arginine residues involved in DNA backbone interactions were mutated to Alanine (Myc RA). While both mutants showed impaired E-box recognition in vitro, their over-expression in murine fibroblasts revealed very different genome-interaction profiles, Myc RA showing no detectable DNA binding, and Myc HEA retaining about half of the binding sites seen with Myc wt. The analysis of the binding intensity of Myc wt and Myc HEA at their binding sites revealed that, as expected, Myc wt bound more strongly the sites containing the E-box, while Myc HEA bound the sites with an E-box as well as the sites without it, confirming that the mutant lost the sequence-specific recognition ability. The interactions retained by the Myc HEA were dramatically reduced with the protein expressed from the endogenous c-myc locus, though genome engineering. Thus, unlike Myc RA, the Myc HEA mutant retained non-specific interactions with genomic DNA (detectable at elevated protein levels) but failed to engage more stably through sequence-specific DNA contacts. In spite of this residual DNA-binding activity, Myc HEA was profoundly impaired in its biological function, undistinguishable from Myc RA: in particular, neither mutant could substitute for wild-type Myc in supporting cell proliferation in murine fibroblasts, whether at normal or supra-physiological levels. While the assessment of transcriptional activities is still ongoing, we conclude that E-box recognition is essential for Myc’s biological function.

One of the major limits of current therapies against cancer and viral infections is the nonspecific toxicity that they often cause on healthy tissues because of their impact on important cellular mechanisms shared, to different extents, between diseased and healthy cells. For this reason, there is an unmet need for more specific and more effective therapies. Wherefore, the aim of my project is the development of a novel strategy, with potential for therapy, that allows the induction of sequence-specific DNA lesions (DNA double-strand break, DSB), by the use of the CRISPR/Cas9 system targeting a genome sequence abnormality in diseased cells, while sparing normal cells. Potential applications of this approach can be cancer cells carrying genomic mutations or chromosomal rearrangements and infected cells carrying an integrated proviral genome. Importantly, whether the aberrant genome sequences are expressed or not is irrelevant for the efficacy of this approach. As a proof of principle, I generated, in two parallel cell systems, an isogenic pair of cell lines with a healthy and a diseased counterpart. The “diseased” target sequence is an integrated proviral genome. To generate them, I infected HeLa and RKO cells with a lentiviral vector containing the sequence of the green fluorescent protein (GFP). I then treated these two cell systems with the purpose of inducing a DSB by retroviral transduction of the Cas9 endonuclease and its RNA guide targeting the integrated GFP sequences. As a negative control, I treated these cell lines in parallel with a Cas9 carrying a scramble guide that does not recognize any sequence in the human genome. Upon these treatments, I observed a preferential reduction of proliferation and an increased mortality in cells bearing the target sequence and transduced with the targeting RNA guide compared to cells without the target sequence or transduced with the scramble guide. I also observed that Cas9-mediated DNA damage is associated with the formation of micronuclei which often stain positive for cGAS and activate an inflammatory response. These results suggest the possibility to “weaponize” the CRISPR/Cas9 system for the elimination of cells with an aberrant genome. However, cells can survive DNA damage insults by repairing them. In order to address this mechanism of “resistance” to the treatment, I investigated if the generation of a sequencespecific DSB can be combined with the inhibition of its repair. Indeed, Cas9-induced DNA damage and inhibition of DNA repair by non-homologous end-joining (NHEJ) by the use of a pharmacological inhibitor of the DNA-dependent protein kinase (DNA-PK), a DNA repair factor involved in NHEJ, further kill target cells. However, DNA-PK inhibition lacks sequence specificity in its activity, thus impacting on the repair of endogenous DNA damage too. For this reason, a sequence-specific DSB repair inhibitor would be desirable. Our group has previously demonstrated that DSBs trigger the recruitment of RNA polymerase II that generates damage-induced long non-coding RNAs (dilncRNAs) at DSB. DilncRNAs are the precursors of small non-coding RNAs called DNA damage response RNAs (DDRNAs) and the interaction between dilncRNAs and DDRNAs is necessary for the recruitment of the proteins involved in DDR, including DNA repair. Noteworthy, antisense oligonucleotides (ASO) against these damage-induced RNA species impair their functions and inhibit the assembly of DDR factors in the form of foci and thus they are effective sequence-specific DNA repair inhibitors. In cells treated with Cas9, I observed a reduction in DDR foci, compared to controls, upon treatment with sequence-specific ASO, confirming the efficacy of ASO also in my experimental system.

When genome integrity is perturbed, surveillance and repair mechanisms are activated to restore genome integrity through high fidelity DNA repair. However, in some physiological situations, those mechanisms are channeled away from integrity towards mutations and recombinations. During the diversification of the immunoglobulin locus in B cells, Activation Induced Deaminase (AID) triggers the physiological introduction of mutations. The current work was based on the observation that upon simple lesion generation, AID-induced deamination of a cytosine to uracil, the resolution by the molecular mechanism of DNA repair can lead to different outcomes. This homeostatic outcome, error-free or error-prone, is governed by specific cellular context and processes associated with DNA. To uncover the regulation of the pathway choice an in vitro system, named in vitro resolution (IVR), was developed. In the 1st phase of the IVR, AID was targeted to a DNA plasmid for uracil lesion generation. In the 2nd phase, a cellular extract resolved the lesions via Base Excision Repair [BER, divided in short patch (SP)-BER or long patch (LP)-BER] or Mismatch Repair (MMR). The quantitative nature of the IVR provided a novel means to precisely quantitate the contribution of each single DNA repair pathway. This set-up allowed us to evaluate how different cellular environments influenced the choice. Cell origins presented quantitative differences in DNA repair kinetics: a) overall sensitivity, b) non-B cells activating non-canonical MMR first, c) B cells activating SP-BER first, and d) LP-BER is significantly activated only in B cells. To understand the possible molecular mechanisms, we analysed single components known to influence DNA repair, such as transcription, protein availability, and chromatin. Changing the DNA substrate to either prefer or avoid forming nucleosomes, we uncovered significant changes in AID deamination preference and in DNA repair pathway choice. DNA with nucleosome favourable base-stacking preferred LP-BER, while non-nucleosome stacked DNA preferred SP-BER and MMR. Overall our findings provide novel insight into the cellular context that can influence DNA repair. The use of B cells and cancer cell lines can recapitulate in vivo Ig locus diversification, and our findings have a direct bearing in understanding mechanisms of tumorigenesis.

Pharmacogenomics is the study of how genes affect a individual response to drugs to develop medications tailored to a person’s genetic makeup because the efficacy/safety profile have not be the same way for everyone. Mitochondria are characterized by a unique milieu, with an alkaline and negatively charged interior (pH value 8) due to the proton pumping associated with OXPHOS and a series of specific channels and carrier proteins. As a consequence, mitochondria can easily accumulate lipophilic compounds of cationic character and weak acids in their anionic form, particularly amphiphilic xenobiotics including ethidium bromide, 1-methyl-4 phenylpyridinium (MPP+), paraquat (1,1’-dimethyl-4,4’-bipyridinium dichloride; PQ) and others that can penetrate the inner mitochondrial membrane (IMM) freely since in their undissociated forms. Indeed, it is well understood that many drugs and chemicals can cause mitochondrial dysfunction (mitotoxicity) by interacting with mitochondrial DNA (mtDNA), protein synthesis, respiratory chain, other metabolic processes, channels and transporters Moreover, due to its peculiar uniparental maternal inheritance and high mutation rate, mtDNA presents different clusters of population-specific-polymorphism (SNPs) that characterize different maternal lineages (mitochondrial haplogroups). It has been demonstrated that many non-synonymous SNPs, cause amino acid variations in the mitochondrial-encoded proteins, potentially modifying OXPHOS activity and ROS production. Some of these haplotypes may confer vulnerability to, or protection from, various common diseases. Well-documented examples are the role of mtDNA haplotype in Parkinson’s disease (PD) and Leber’s hereditary optic neuropathy (LHON). It has been proposed that European haplogroups J and K are protective for PD. On the other hand the haplogroups J and T may influence mitochondrial dysfunction, resulting in an increased risk of PD. In addition the 11778/ND4, 14484/ND6 and 3460/ND1 LHON mutations are associated respectively with mitochondrial subhaplogroup J2b, J1c and K, as these mtDNA backgrounds may increase penetrance of LHON mutations. Several reports suggest that environmental factors such as pesticides (e.g. rotenone), herbicides (e.g. paraquat) and MPTP or 1-methyl 4-phenyl 1,2,3,6-tetrahydro-pyridine (contaminant in the illicit synthesis of opiates) increase the risk of PD due to a reduction in ATP synthesis and increase of reactive oxygen species (ROS). Furthermore, tobacco smoking has been proposed as an environmental trigger of visual loss in LHON, due to the presence of substances, contained in the tobacco that can directly act inhibiting CI. Researchers have also associated non synonymous variants in mtDNA with the development of side effects of drugs. Effectively the analysis of mtDNA haplogroup in patients with cancer treated with chemioterapic agent cisplatin (cisPt) revealed an increased incidence of hearing loss in haplogroup J, due to inhibition of mtDNA replication. It has also been shown that patients treated with the antibiotic Linezolid may develop LHON-like optic neuropathy, myelosuppression and lactic acidosis. This is possibly due to the inhibition of mitochondrial protein synthesis, which is modulated by the SNPs at positions 2706 and 3010, in the 16S gene of mtDNA. This sequence region is predicted to be very close to the Peptidyl Transferase Center (PTC) that is the binding site of several antibiotics. To demonstrate that mitochondrial genetic variability may influence individual susceptibility to drugs toxicity (Linezolid and CisPt) or to toxic environmental factors (rotenone, MPP+, paraquat and cigarette smoking) we assessed in vitro cell viability, mitochondrial functions including ATP synthesis, activity of OXPHOS complexes and ROS generation, and biogenesis (mtDNA copy number) in a collection of transmitochondrial cytoplasmic hybrids (cybrids) carrying divergent human mtDNA haplogroups (N1b, H, J, T, U, and K) or LHON mutations, that have been defined by sequencing of D-loop region and then of the entire mtDNA. Cybrids were constructed from fibroblasts obtained, after informed consent, from skin biopsies of unrelated healthy subjects and LHON patients. The results of this study demonstrated that mitochondrial genetic variability may influence individual susceptibility to drugs or environmental factors toxicity, highlighting interesting associations between specific haplogroups, mitochondrial functional alterations, and toxic agent. More in details: 1) haplogroup K1 was found to play a protective role against rotenone toxicity, whereas haplogroup J1 seem to be more susceptible to the action of both rotenone and MPP+; 2) haplogroup T seems to be more susceptible to the action of paraquat; 3) haplogroups H12 and T1 in association with the LHON mutation 3460/ND1, and haplogroups J1c and J2A all increase the susceptibility to mitochondrial damage after smoke exposure. Moreover haplogroup H1, characterized by SNPs 2706A/3010A in 16SrRNA is the most sensitive to Linezolid toxicity and haplogroup J appears to act as risk factor in CisPt toxicity. Even though future studies will be necessary to better understand the mechanism of action of some of these molecules, studying the association between mitochondrial haplogroup and toxicity of drugs and chemicals is extremely useful to prevent toxicity in predisposed subjects. This may avoid the occurrence of adverse reactions leading to the withdrawal of drugs from the market or Black Box warnings by FDA. For these reasons, pharmaceutical companies have introduced early in the drug-development process stringent in vitro studies to evaluate mitochondrial function (respiratory chain, ROS, membrane potential and mtDNA).
La farmacogenomica si occupa di indagare gli effetti di un determinato farmaco o sostanza chimica in base al genotipo dell’individuo con lo scopo di personalizzare le cure e fornire le terapie adeguate. I mitocondri presentano un potenziale di membrana (Δψ) di 180mV, una matrice alcalina (pH 8) con carica negativa e possono accumulare al loro interno sia sostanze cariche positivamente sia acidi deboli in forma anionica, rendendosi bersaglio primario o secondario dell’azione di farmaci e agenti tossici. I mitocondri sono dotati di un proprio corredo genomico (mtDNA) che si caratterizza per un’ereditarietà uniparentale materna un elevato tasso di mutazione e numerose varianti genetiche, distinte in aplogruppi. È noto che variazioni non sinonime nel mtDNA causano alterazioni funzionali di proteine implicate nel processo OXPHOS, tali da supportare studi per comprendere se la variabilità mitocondriale possa svolgere un’azione protettiva o rappresentare un fattore di rischio per l’insorgenza di patologie. In particolare è stato dimostrato che soggetti appartenenti agli aplogruppi J e K e con polimorfismo 10398G nel gene ND3 (CI) si correlano a minore rischio di sviluppare il Morbo di Parkinson (PD). Al contrario la variante 4216C nel gene ND1 (CI), comune al sottogruppo JT, parrebbe correlata a un aumentato rischio di malattia. Inoltre è stato dimostrato un maggiore rischio di Neuropatia Ottica di Leber (LHON) se le mutazioni 11778/ND4 (CI), 14484/ND6 (ND1) e 3460/ND1 (CI) si associano agli aplogruppi J2b, J1c e K rispettivamente. Ulteriormente studi ipotizzano che agenti inquinanti ambientali quali pesticidi (es. rotenone), erbicidi (es. paraquat) e MPTP o 1-metil 4-fenil 1,2,3,6-tetraidro-piridina (composto secondario contaminante nella sintesi illecita di oppiacei) siano causa di un maggiore rischio di PD per un’alterata funzionalità mitocondriale con riduzione della sintesi di ATP e aumento di specie reattive dell’ossigeno (ROS). In altri studi s’ipotizza che il rischio di perdita della vista in carrier di mutazioni LHON, aumenta se il soggetto è fumatore, per azione sul CI della catena respiratoria. Infine ancora dati di letteratura associano numerose variazioni mitocondriali non sinonime allo sviluppo di patologie collaterali a trattamenti farmacologici. E’ stato dimostrato che soggetti appartenenti all’aplogruppo J, presentano un aumento del rischio di sviluppare ototossicità dopo trattamento con l’antitumorale Cisplatino (CisPt) per inibizione della replicazione del mtDNA mentre pazienti con SNPs nella regione 16SrRNA in posizione 2706A e 3010A trattati con l’antibiotico Linezolid, sviluppano mielosoppressione, neuropatia e acidosi lattica in seguito a inibizione della sintesi proteica mitocondriale. L’attività di ricerca ha avuto come oggetto di studio il ruolo della variabilità del genoma mitocondriale (mtDNA) nella suscettibilità individuale alla tossicità da farmaci (Linezolid e Cisplatino) o da agenti tossici inquinanti ambientali (rotenone, MPP+, paraquat ed estratto di fumo) in un modello in vitro costituito, da ibridi transcitoplasmatici (cibridi) creati mediante fusione di fibroblasti da donatore, dopo enucleazione, con cellule di osteosarcoma private di mtDNA (rho0). Sono così stati originati cloni di cibridi appartenenti ai principali aplogruppi mitocondriali europei (N1b, H, J, T, U, K) o portatori di diverse mutazioni LHON, e caratterizzati mediante analisi di sequenza della regione non codificante di 1122 bp o“Displacement loop” (Dloop) e dell’intero genoma mitocondriale. Gli studi sono stati eseguiti valutando la vitalità cellulare, la funzionalità (sintesi di ATP, produzione di ROS) e la biogenesi mitocondriale (numero di copie di mtDNA). I risultati ottenuti da queste analisi hanno dimostrato che una variabilità genetica mitocondriale può influenzare la suscettibilità individuale alla tossicità da agenti tossici inquinanti ambientali e farmaci. In particolare sono state evidenziate alcune interessanti associazioni tra specifici aplogruppi mitocondriali, alterazioni mitocondriali e agente tossico: 1) l’aplogruppo K1 sembra svolgere un’azione protettiva rispetto al rotenone mentre l’aplogruppo J1 sembra più sensibile all’azione del pesticida e del MPP+ anche se quest’ultimo è meno specifico e affine al CI.; 2) l’aplogruppo T sembra più suscettibile all’azione del paraquat.; 3) gli aplogruppi H12 e T1 quando associati a mutazione LHON 3460/ND1 e gli aplogruppi J1c e J2a aumentano la suscettibilità al danno mitocondriale da fumo. Successive evidenze hanno dimostrato che l’aplogroup H1, caratterizzato dai polimorfismi 2706A e 3010A nel gene 16SrRNA, sembra essere il più sensibile all’azione tossica del Linezolid così come l’aplogruppo J è risultato più sensibile alla citotossicità da Cisplatino. Questi dati suggeriscono che procedere nello studio di associazioni tra aplogruppo mitocondriale e tossicità da farmaci e agenti chimici potrebbe consentire di individuare precocemente il rischio tossicologico individuale. Queste conoscenze potrebbero essere di particolare impatto per prevenire reazioni avverse a farmaci in alcuni casi causa di ritiro dal commercio o black box. Con questo scopo di recente le aziende farmaceutiche hanno introdotto nelle fasi iniziali dello sviluppo di un farmaco studi in vitro per valutare eventuali effetti sulla funzionalità mitocondriale (catena respiratoria, ROS, potenziale di membrana e mtDNA).

FUS (anche nota come TLS) è una RNA-binding protein espressa in maniera ubquitaria nelle cellule umane, che è stata descritta essere fusa a fattori di trascrizione in alcuni sarcomi ed è presente in aggregati proteici nei neuroni di pazienti affetti da forme ereditarie di Sclerosi Laterale Amiotrofica. FUS è stata coinvolta in numerosi processi cellulari, come l’espressione genica, la regolazione trascrizionale, il pre-mRNA splicing, e il processamento dei miRNA. In più, è stata coinvolta nella risposta al danno al DNA e in particolare è stato dimostrato che rilocalizza ai siti di danno nelle fasi precoci della risposta. Inoltre, a seguito di danno al DNA, FUS agisce come SUMO E3 ligasi per la proteina EBP1, mediandone la sumoilazione, una modifica post-traduzionale necessaria alla sua attività onco-soppressiva. L’esatta funzione di FUS in questi processi non è ancora stata chiarita, quindi, per elucidare la sua attività abbiamo studiato il suo interattoma, in cellule umane, in condizioni basali e a seguito di stress genotossico. Gli interattori di FUS sono stati isolati tramite immunoprecipitazione su estratti proteici totali di cellule HEK293T esprimenti FUS-flag ricombinante, in tre condizioni diverse: con e senza trattamento degli estratti con RNAsi e con trattamento e lavaggio ad alta concentrazione salina. Gli interattori sono stati identificati tramite spettrometria di massa, con i seguenti risultati: 546 proteine nei campioni non trattati, 134 nei trattati, 53 nei trattati con alto sale; 40 proteine sono state identificati in comune a tutte e tre le condizioni e possono essere considerate gli interattori di FUS più conservati. Questi ultimi sono proteine annotate soprattutto a processi biologici associati all’RNA, in linea con i ruoli già descritti di FUS nel metabolismo dell’RNA; in questo set di proteine sono presenti HNRNPs, RNA elicasi, fattori di splicing, e proteine regolatorie della trascrizione. In particolare, tra gli interattori più conservati, c’è un arricchimento in proteine facenti parte del complesso spliceosomale minore di tipo U12. L’interazione di FUS con queste proteine è stata validata ed esperimenti funzionali di splicing hanno dimostrato che FUS lega gli introni di tipo U12 aumentandone lo splicing. Inoltre, sono stati identificate tra gli interattori di FUS proteine coinvolte nella risposta e riparazione dei danni al DNA (DDR), tra cui Ku70, Ku80, PSF, p54, PARP1, e altre. Pertanto, per caratterizzare il ruolo di FUS nella DDR è stata condotta un’analisi di interattoma a seguito di stress genotossico. Cellule HEK293T esprimenti FUS-flag sono state trattate con Etoposide per generare rotture a doppio-filamento del DNA ed gli interattori identificati a seguito di immunoprecipitazione e spettrometria di massa quantitativa. L’analisi ha restituito una lista di interattori la cui affinità per FUS era aumentata o diminuita a seguito del danno. In particolare, la maggior parte delle proteine con un’aumentata affinità hanno la capacità di legare gli acidi nucleici, in particolare l’RNA. Recentemente, le RNA-binding proteins, sono state descritte come i principali regolatori della DDR. Inoltre, alcune di queste sono state descritte come sumoilate e ciò potrebbe suggerire un ruolo di FUS come E3 ligasi nella DDR. Tuttavia, l’esatto ruolo di FUS nella DDR deve ancora essere chiarito, e i nostri risultati suggeriscono che potrebbe giocare un ruolo nella regolazione dell’espressione dei geni delle proteine coinvolte nella DDR, tramite la sua attività di splicing, o funzionare come proteina di supporto per reclutare RNA-binding proteins al sito di danno al DNA.

Cells have evolved several DNA repair mechanisms to maintain their genetic information unaltered and a DNA damage response pathway (DDR) coordinates DNA repair with several cellular events including a cell-cycle arrest until damaged DNA is repaired. When cells fail to repair DNA lesions, they undergo either apoptosis or cellular senescence. Tools commonly used to detect DNA lesions rely on indirect, antibody-based recognition of proteins associated to DNA breaks. Unfortunately, these tools do not allow direct and precise localization of the breaks, leaving several biological questions unanswered. I validated and optimized BLESS and BLISS, two methods that allow genome-wide single-nucleotide resolution mapping of DNA double strand-breaks (DSBs). Using these techniques, I studied the impact of DSBs on transcription. I characterized a DDR-dependent transcription inhibition around breaks. Differently, I observed that following macrophages LPS-stimulation, a transient wave of DSBs is induced at LPS-specific enhancers and it correlates with their transcription activation, thus suggesting a new mechanism for transcription activation involving controlled DNA damage generation. BLESS and BLISS cannot be applied to single-cell studies. Thus, I developed a new method, named DI-PLA, for the detection and imaging of DSBs in fixed cells and tissues. I applied DI-PLA to demonstrate that senescence cells and aged tissues accumulate DSBs, which are associated with persistent DDR activation, that is known to fuel cellular senescence. Finally, I developed a modification of BLESS to discriminate between DSB bearing telomeres and deprotected telomeres which could be applied to further characterize the mechanisms of DDR activation at telomeres.

DNA Topoisomerases are essentials enzymes in biological process involving the relaxation of the superhelical tension of the DNA molecules. Human DNA topoisomerase I (hTop1p) is a monomeric conserved enzyme of 91 KDa, subdivided into four domains: the N-terminal; the core constituted by three subdomains; the C-terminal domain containing the catalytic residue Tyr723; the linker connecting the core with the C-terminal domain, locating the active site in the catalytic pocket, and characterized by two protruding coiled coil α-helices. DNA relaxation occured with a mechanism of DNA strand rotation in a multiple step process where the 5’-OH end is free to rotate around the intact strand. Two conformational changes are involved in this dynamic process: the enzyme binding the DNA through an open shape, then closed clamp that completely embraces the DNA. No external energy source occurred, but arises from the release of the superhelical tension. In addition hTop1p is of great interest since is the cellular target of the antitumor drug camptothecin (CPT). The reversible binding of the drug to the covalent complex DNA-Top1p brings to lethality due to religation inhibition and double strand breaks formation in a S-phase dependent manner. Different data supported the “controll rotation mechanism” to explain how this enzyme can relax both positevely and negatevely supercoiled substrates. First of all crystal structures [17] of human Top1 with a 22 bp oligonucleotide reported no sufficient space inside the clamp for a free rotation. Moreover a mutant isoform of the protein with 2 cysteines produced opposetely to the lips domains, locked the clamp through a disulfide bridge and reported that an opening conformation was not required for the relaxation activity [21] [135]. Also single-molecule experiments suggested that friction and torque are necessary for the relaxation process and the number of supercoils removed per cleavage-religation cycle is strictly dependent from the number of supercoils of the DNA substrate. The aim of this project is focused on the contribute of key residues located al the level of the hinge and involved in the control rotation mechanism during DNA relaxation. In my experiments Saccharomyces cerevisiae strains were used as a model organism to investigate a series of topoisomerase IB mutants: Pro431Gly; Arg434Ala; Arg434Cys; Trp205Cys. These substitutions are interesting because of their position in the enzyme three-dimensional structure. The flexible hinge (429-436 residues) facilitating clamp opening by a stretching or bending motion pivoting around Pro-431 and the cluster of large aromatic residues around the top of the hinge, probably are involved in controlling the movements of the α-helix and the proper closing of the clamp. In 2005 Sari and Andricioaei simulation analyses reported the possible role of the lips for the relaxation of the positive supercoils and the strechting of the hinge for the negative supercoils removal. Yeast strains lacking for endogenous topoisomerase I gene were transformed with YCpGAL1-hTOP1, YCpGAL1-hTOP1Pro431Gly, YCpGAL1-hTOP1Arg434Cys, YCpGAL1-hTOP1Arg434Ala, YCpGAL1-hTOP1Trp205Cys plasmids and viability was verified in a drug yeast sensitivity assay. Transformants were spotted in presence and absence of camptothecin at different concentrations. Moreover CPT sensitivity for each mutant was analyzed through measures of the stability of the covalent complex formation. Results are congruents and are also supported by Frohlich and Knudsen data, in 2007, suggesting a role for the Trp-205 and surrounding residues in the control of strand rotation during negative but not positive supercoiled removal, and reported a more CPT sensitivity during relaxation of positively versus negatevely supercoils. EKY3 strains expressing htop1W205Cp showed a partial resistance phenotype, and top1R434A, top1R434C mutants are not sensitive to lower drug dose (0.01Le DNA topoisomerasi sono enzimi essenziali in tutti i processi biologici in cui è necessario un riarrangiamento topologico della doppia elica. La DNA topoisomerasi IB umana (hTop1p) è una proteina monomerica di 91KDa, caratterizzata da 4 domini: uno N-terminale; uno “core” costituito a sua volta da 3 subdomini; un C-terminale contenente il residuo catalitico in posizione Tyr723 e una regione linker costituita da 2 α-eliche sporgenti e che connette il dominio core con il C-terminale. Il meccanismo di rilassamento del DNA è un processo multiplo attraverso il quale un filamento di DNA con un’estremità libera al 5’-OH è in grado di ruotare intorno a quello rimasto intatto. L’enzima lega la doppia elica dapprima assumendo una conformazione aperta, e successivamente si richiude attorno al substrato, in questo modo l’energia necessaria per la reazione di topoisomerizzazione è fornita dallo srotolamento della superelica e il rilascio di energia torsionale. hTop1p è anche target specifico di un farmaco antitumorale noto come camptotecina (CPT). Il legame reversibile tra CPT e il complesso DNA-enzima è dipendente dalla fase S del ciclo cellulare e porta ad apoptosi in seguito all’inibizione della fase di riligazione e alla formazione di frammenti a doppio filamento (noti come DSBs double strand breaks). Il meccanismo che spiega come questo enzima sia in grado di rilassare sia superavvolgimenti positivi che negativi è definito “meccanismo di controllo della rotazione” e vi sono diversi dati a suo supporto. La struttura cristallografica della hTop1p in complesso con un frammento di DNA di 22pb, ottenuta nel 1998 da Stewart e i suoi collaboratori ha evidenziato che non vi è spazio sufficiente per una rotazione libera all’interno della “tasca enzimatica”. Inoltre mediante un mutante della hTop1p con 2 residui terminali delle regioni delle “lips” sostituiti da 2 cisteine in modo da ottenere un ponte disolfuro, è stato possibile bloccare l’enzima nella conformazione chiusa e dimostrare comunque l’attività di rilassamento da parte della proteina [21] [135]. Vi sono inoltre esperimenti di “single-molecule” che evidenziano come il numero di superavvolgimenti rimossi per ciclo di taglio e riligazione sia direttamente proporzionale al numero di avvolgimenti del DNA substrato e che durante la topoisomerizzazione si registrano attriti e forze rotatorie derivanti dal meccanismo di rotazione del DNA durante il suo rilassamento. Lo scopo di questo progetto di ricerca verte sull’indagine di residui chiave localizzati nella regione hinge (residui 429-436) e coinvolti in tale meccanismo. Negli esperimenti è stato utilizzato l’organismo modello Saccharomyces cerevisiae, nel quale sono state introdotte le seguenti mutazioni: Pro431Gly; Arg434Ala; Arg434Cys; Trp205Cys. Tali sostituzioni risultano di grande interesse per quanto concerne la struttura tridimensionale: il residuo 431 caratterizzato da una prolina si è ipotizzato essere coinvolto in movimenti flessori e di stretching dell’hinge attraverso cui l’enzima medierebbe l’apertura/chiusura e l’interazione con i residui aromatici situati nel suo intorno. Sari e Andricioaei nel 2005 attraverso studi di dinamica molecolare evidenziarono il contributo della regione delle lips nel rilassamento dei superavvolgimenti positivi, mentre riportarono uno stretching dell’hinge per quelli negativi. Dapprima sono stati utilizzati ceppi di lievito deleti della topoisomerasi I endogena per trasfettare i plasmidi con all’interno la sequenza di espressione per la hTop1p mutata ed è stata analizzata la capacità di formare colonie nonché la loro vitalità in presenza di camptotecina a diverse concentrazioni. La sensibilità al farmaco è stata inoltre analizzata mediante saggio di equilibrio taglio-riligazione e valutando la formazione dei complessi di taglio per ciascun mutante in presenza e assenza di camptotecina. I risultati si sono rivelati congruenti con quanto già riportato nel 2007 dal laboratorio di Knudsen, secondo cui il residuo Trp205 e l’intorno aromatico regolano il meccanismo di rotazione durante il rilassamento di superavvolgimenti negativi e riportando una maggior sensibilità al farmaco durante la rimozione di substrati positivi piuttosto che del segno opposto. Il mutante htop1W205Cp nei miei esperimenti ha mostrato un fenotipo parzialmente resistente mentre per i mutanti top1R434A e top1R434C si è riportata una maggior sensibilità e capacità di formare colonie a dosi inferiori a 0.5ug/ml. Per ciascuna proteina è stata misurata la sua attività catalitica nel rilassare superavvolgimenti positivi e negativi, in condizioni di processività e distributività a forza ionica bassa (50 mM), a 150 mM (condizione ottimale) e a forza ionica elevata (500 mM e 1000 mM). In questo modo è stato possibile valutare l’affinità di legame di ciascun mutante per il DNA ed evidenziare aspetti biochimici funzionali legati alla mutazione specifica. Per quanto riguarda il mutante top1R434A è stata riportata attività catalitica ad alte concentrazioni saline, fino a 1000 mM KCl, ed è stata misurata una velocità maggiore nel rilassare substrati positivi piuttosto che negativi, e in ogni caso più performanti sia rispetto l’enzima wild type che gli altri mutanti. La sostituzione dell’arginina (R434) con un’alanina ha migliorato l’affinità di legame per il DNA dell’enzima e questo è stato ancor più evidenziato da saggi di rilassamento in cui top1R434A mostrava capacità di rilassamento, a basse e alte forze ioniche, di due DNA topologicamente diversi, ma contemporaneamente presenti nella miscela di reazione. L’enzima è perciò in grado di staccarsi e riassociarsi ad una nuova molecola di DNA. Inoltre tramite analisi di modelling molecolare è stato possibile indagare il contributo reale dei diversi domini della proteina wild type a confronto con quella mutata nel residuo R434, durante il processo di topoisomerizzazione.

DNA replication is a fundamental macromolecular event that is essential for cell division. During each cell cycle the entire genome has to be precisely duplicated to ensure genome integrity and stability. In the process of DNA replication, replication forks encounter endogenous and exogenous lesions and these lesions have to be rectified to transfer stable genetic material to daughter cells. Emerging evidences connected a role for Homologous Recombination (HR) proteins to replication fork protection during unperturbed DNA replication. Since HR protein, BRCA2 and Rad51 are essential for cell survival we used Xenopus laevis cell-free extract to dissect the function of BRCA2 and Rad51 during chromosomal DNA replication. Using Electron Microscopy (EM) we show that BRCA2 and Rad51 function together to prevent single-stranded DNA (ssDNA) accumulation at and behind the forks during unperturbed DNA replication. We further discovered that BRCA2 mediates interaction between Rad51, Polymerase alpha (α) and Polymerase delta (δ) and this interaction likely promote efficient re-priming and polymerising activity at stalled replication forks to prevent ssDNA accumulation. Moreover we show that inhibition of replicative polymerases in the absence of Rad51 results in increased frequency of replication fork reversal activity. We further found that replication fork reversal is predominantly induced by an annealing helicase SMARCAL1 in the absence of Rad51. Collectively our findings indicate that, to prevent ssDNA accumulation and aberrant replication fork architecture, timely re-priming of Polymerase alpha mediated by Rad51 is essential. Hence, loss of Rad51 impact replication fork architecture, eventually resulting in chromosomal abnormalities.

The DNA damage response (DDR) coordinates DNA repair events and transiently arrests cell-cycle progression until DNA damage has been removed. If the damage is not resolved, cells can enter an irreversible cell cycle arrest called cellular senescence. In irradiation-induced senescent cells a large fraction of persistent DDR markers are associated with telomeric DNA, both in cultured cells and in in vivo tissues. The aim of my PhD project was to investigate the mechanism underlying this phenomenon. I showed that persistent DDR activation has a causative role for the senescence-associated cell cycle exit and that a double-strand break (DSB) within telomeric repeats is inducing a more protracted DDR activation compared with a non-telomeric one in human cells. The DDR persistency at telomeres is neither dependent on their heterochromatic state nor on TRF2 loss from telomeres during senescence establishment. Rather, TRF2 recruitment next to a DSB, in the absence of telomeric DNA, is sufficient to induce a more protracted site-specific DDR focus and to impair DSB repair in mouse cells. Ageing is associated with accumulation of markers of DDR activation. In terminally differentiated brain neurons from old primates, I observed DDR activation at telomeres that were not critically short. Taken together, these results strongly suggest that TRF2 inhibits DNA repair at broken telomeres, contributing to the accumulation of unrepaired, DDR-positive telomeres during ageing. This can in turn trigger cellular senescence and impair tissue homeostasis providing a mechanism for ageing also in non-proliferating tissues. Finally, I focused my attention on DICER and DROSHA-dependent DNA damage response RNAs (DDRNAs), novel components of the DDR machinery, which have been described to be necessary for DDR activation at DSBs. I showed that RNase A treatment as well as DICER or DROSHA down-regulation impair DDR activation at uncapped telomeres and that DICER and DROSHA may have a role in chromosomal fusions. Furthermore, in cells with dysfunctional telomeres, the inhibition of telomeric DDRNAs using inhibitory oligonucleotide molecules with a complementary sequence can prevent DDR activation and senescent-associated cell cycle arrest. These data indicate that at uncapped telomeres, DDRNAs with telomeric sequences are generated and that they are necessary for DDR activation and chromosomal fusions.

Eukaryotic cells have evolved the ATR/hCHK1, MEC1/RAD53 kinase-mediated signal transduction pathway, known as replication checkpoint, to protect and stabilize stalled replication forks in human cells and budding yeasts, respectively. rad53 mutants, exposed to high doses of the DNA replication inhibitor hydroxyurea (HU), accumulate hemireplicated, gapped and reversed forks, while treatments with low HU doses induce massive chromosome fragmentation. The aim of my work was to better understand the molecular mechanisms through which Rad53 prevents unusual alterations of the architecture of the stalled replication forks and chromosome fragility, under replication stress. We revealed that Rrm3 and Pif1, DNA helicases assisting fork progression across pausing sites in unperturbed conditions, are detrimental in rad53 mutants experiencing HU-induced replication stress. Rrm3 and Pif1 ablation synergistically rescues cell lethality, chromosome fragmentation, replisome dissociation, fork reversal and ssDNA gaps formation at the forks of rad53 cells exposed to replication stress. We provide evidence that Pif1 and Rrm3 associate with stalled DNA replication forks and are regulated through Rad53-mediated phosphorylation. Our findings uncover a new replication-stress-induced regulative loop in which Rad53 down regulates the Pif1 DNA helicases at the stalled replication forks. In the second part of this thesis we examined the crosstalk between Rrm3, Pif1, the mediator of the DNA damage checkpoint Rad9 and the nuclease Dna2, during unperturbed DNA replication. The experimental evidence collected in this second part of the project, together with pioneering work previously reported from other laboratories, strongly suggests that Dna2, Pif1 and Rrm3 cooperate to finalize late stages of DNA replication.

Alcohol addiction is thought to depend on molecular and cellular adaptations in the brain that result from chronic drug exposure (Fitzgerald and Nestler, 1995 ). This suggests that chronic alcohol abuse involves stable changes in the brain at the molecular and cellular levels responsible for long-lasting alterations in behavior. One of the candidate molecules involved in such mechanisms is brain-derived neurotrophic factor (BDNF). Aberrant regulation of BDNF has been implicated in the development of psychiatric disorders, including schizophrenia, depression, anxiety and alcohol addiction (Moonat et al., 2010). Multiple actions of ethanol on BDNF gene expression and signalling have been described. Nevertheless the considerable complexity within the BDNF gene itself and the multiple mRNAs encoded by up to 9 potential exons is further complicated because of its epigenetic regulation by methylation of BDNF promoters. The knowledge of the promoter that may be differentially regulated during various states of ethanol exposure and a detailed analysis of the effects of different ethanol exposure treatments on mRNA expression is required to understand the biological basis of addiction. This will lead to more effective treatments and eventually to cures and preventive measures to treat alcohol addiction. The aim of this work was to evaluate the effects of ethanol on CpG islands of BDNF exon IX gene in regulating its expression, both “in vivo” and “in vitro”. Rat cerebellar granule cells in culture were exposed to acute ethanol, chronic ethanol or ethanol withdrawal. Our results demonstrate that ethanol exposure increases, the abundance of BDNF exon IX transcript in the three different treatment conditions. We then tested the ability of acute ethanol to alter exon IX methylation by using MSP PCR. Similarly to the effect induced by the two DNA methylation inhibitors, zebularine and RG-108, exposure of cultured neurons to 100 mM ethanol for 3h significantly increased the unmethylated state of BDNF exon IX that was about 2.5 folds grater than the methylated state. This epigenetic action of ethanol was observed also in the hippocampus of male rats treated with increasing doses of ethanol (0.8; 1.6 or 3.0 g/kg; i.p.) tested 1; 3 and 5h after injection. Ethanol induced a significant time and dose dependent increase in BDNF exon IX unmethylated DNA levels, compared with control, which showed a positive correlation with BEC. The increased unmethylated state was accompanied by a corresponding increase in the abundance of BDNF exon IX transcript. The increase in mRNA was dose dependent with the maximal effect at 3.0 g/kg 3h after injection. These results provide the first evidence for an alternative way of ethanol to alter BDNF gene expression. Thus, ethanol induced changes in CpG DNA methylation of BDNF gene seems to be an additional mechanism to implement homeostatic protective actions to prevent adverse effects of ethanol. The ability of ethanol to induce up regulation of BDNF may play a pivotal role and could result from a number of intracellular responses that include the epigenetic mechanism here described. Fitzgerald and Nestler, (1995). Clinical Neuroscience 3(3):165-73. Moonat et al., (2010). Cellular and Molecular Life Sciences 67(1):73-88.

Saccharomyces cerevisiae cells with a single double-strand break (DSB) activate the ATR/Mec1-dependent checkpoint response as a consequence of extensive ssDNA accumulation. The recombination factor Rdh54, a member of the Swi/Sfn2 family of helicase-like proteins, has been implicated in chromatin remodeling and is required for adaptation from a G2/M arrest induced by an unrepaired DSB. Here we show that both the ATR/Mec1 and Chk2/Rad53 kinases phosphorylate Rdh54 protein in the presence of DNA damage, suggesting that the protein is regulated during the DNA damage response. ATR/Mec1 activity is required for Rdh54 localization to a DSB. We will present additional genetic and biochemical evidences on the physiological role of the phosphorylation modification and the role of Rad51 in this. Our results will be considered in the context of cellular regulatory networks that coordinate the checkpoint response and recombination process in the presence of DSB lesions.

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of a covalent enzyme-DNA intermediate, which is reversibly stabilized by the anticancer agent camptothecin (CPT). Crystallographic studies of the 70-kDa C terminus of human Top1p bound to duplex DNA describe a monomeric protein clamp circumscribing the DNA helix. The structures, which lack the N-terminal domain, comprise the conserved clamp, an extended linker domain, and the conserved Cterminal active site Tyr domain. CPT bound to the covalent Top1p-DNA complex limits linker flexibility, allowing structural determination of this domain. We previously reported that mutation of Ala653 to Pro in the linker increases the rate of enzyme-catalyzed DNA religation, thereby rendering Top1A653Pp resistant to CPT (Fiorani, et al., 2003;). Molecular dynamics studies suggested mutation-induced increases in linker flexibility alter Top1p catalyzed DNA religation. However, despite a wealth of biochemical, structural and modeling data on Top1p structure and activity, there is little direct evidence of linker domain flexibility influencing the geometry of the active site. To address this question we asked if the enhanced rate of DNA religation, imparted by the A653P linker mutation, would suppress the DNA religation defect induced by the T718A active site mutation. Indeed, here we describe that the combination of the two mutations (in Top1A653P/T718A), abolished the lethal phenotype of yeast cells expressing the single T718A mutant. The double mutant enzyme was catalytically active in vitro and in vivo, yet was resistant to CPT. Taken together these data indicate long range communications between the flexible linker domain and the active site of the enzyme. The specific activity of the double mutant was decreased in vivo and in vitro, consistent with a decrease in DNA binding. These findings support a model where changes in the flexibility or orientation of the linker alter the geometry of the active site and thereby the kinetics of DNA cleavage/religation catalyzed by Top1p. The X-ray crystal structure of the enzyme covalently joined to DNA and bound to the CPT analog Topotecan suggests that there are two classes of mutations that can produce a CPT-resistant enzyme. The first class includes changes in residues that directly interact with the drug, whereas a second class alters interactions with the DNA and thereby destabilizes the drug binding site. The Thr729Ala, that is part of a hydrophobic pocket in the enzyme C-terminal domain, belongs to a third group of mutations that confer CPT resistance, but do not interact directly with the drug or the DNA. The Thr729Ala mutation has been firstly identified to impart drug resistance on human topoisomerase I in the CPT-11 (Irinotecan, a CPT analog) resistant human lung cancer cell line, PC-7/CPT (Kubota et al., 1992) but our data reveal that the equivalent effect with CPT can not be observed if the enzyme, harboring the same mutation, is expressed in the yeast Saccaromyces cerevisiae. Interestingly, looking at the protein structure, Thr729 seems to be too distant to contact the drug directly and it is not clear how the structure or stability of the intercalation drug-binding pocket is affected by the mutation. The Thr729 resides in the hydrophobic core of the Cterminal domain 12.4 Å from the catalytic tyrosine and 13.1 Å from Asn722 that establishes a water-mediated contact directly to the drug. Changing the Asn722 to Ser, shortening the side chain, is sufficient to impart CPT resistance to the protein (Fertala et al., 2000). According to these data, Redinbo and co-authors assumed that the basis of the Thr729 mutants CPT resistance come from the destabilization of the C-terminal region that could lead to the specific shift in position of Asn722, consequentially inducing the breakage of the water-mediated contact with the drug (Chrencik, et al., 2004). In order to confirm these hypotheses, we analyzed the effect of Thr729 substitutions to Lys, Pro and Glu on the in vivo and in vitro catalytic activity and drug sensitivity of the human DNA topoisomerase I. These three substitutions were chosen for their positive or negative charge, Lys and Glu, respectively, and for their capability in distorting the a-helix as in the case of Pro. Our results show that the 729 position is a key point in maintaining the correct geometry of the hydrophobic pocket of the C-terminal domain. In fact, even if the enzyme keeps on its catalytic properties and its sensitivity to CPT in the presence of a Thr729Ala mutation, a dramatic CPT resistance effect could be observed when the Thr729 was mutated to Lys or Glu, and a minor consequence could be seen in the presence of a Pro. Furthermore, the Thr729Glu mutant shows a remarkable defect in the DNA binding indicating that the integrity of the C-terminal geometry is essential for the preservation of the correct interactions between the enzyme and its substrate during the progression of the catalytic cycle. Dynamic simulation experiments propose a structural and dynamical interpretation for the role played by residue 729 in these long-range protein–DNA communications (Chillemi et al. 2008 ). The Thr729 is located in the C-terminal domain and its side chain forms a 2.6-A? hydrogen bond with the hydroxyl group of Tyr619. This interaction stabilizes the contacts between the Cterminal and core subdomain III regions of the CAT, that extends from the top half of the molecule downward by a couple of long helices (8 and 9) functioning as an hinge that opens and closes the enzyme around DNA. The most complete structured Nterminal domain of the topoisomerase I protein runs from residue Ile215 to Gly201. These fifteen amino acids pack against part of core subdomain I, the C-terminal domain, and the putative hinge region of the core subdomain to form a hydrophobic cluster. Notably, the cluster is well conserved among eukaryotic type IB topoisomerases (Redinbo et al., 2000). This stretch of N-terminal domain interacts with the ?-helix that connects the two lobes of the protein; specifically, the Trp205 is closer to Arg434, located at the top of “connecting” helix. Rising from these observations, the N-terminal domain positioning is thought to play an important role in the upper portion of this helix that is considered as an hinge involved in the opening and clamping crucial movement in the topoisomerase catalytic process; this portion is also protease sensitive only in the absence of the DNA (Stewart et al., 1996). The residue Pro431 is located in the upper portion of hinge region; where is situated a bent loop . The presence of this structure could be caused by this residue. In order to verify the function of proline 431 we realized a Pro431Gly mutant, introducing a residue lacking of side chain. In this way is possible remove the structural constrictions imposed by proline residue. To clarify the role of interactions between Nterminal domain and hinge region we have been realized all mutants in full length and Topo70 versions. To investigate the cluster of interactions between Arg 434 and Trp 205 the residue Arg 434 is mutated in Ala and Cys. These two substitutions were chosen for their capability in modifying the charge and structure of region in the Ala case and for capability in making a covalent bound in the double mutant (W205CR434C) in the case of Cys. The hinge mutant are lethal when express in yeast system. The lethality is not influenced by presence of N-terminal domain , in fact the topo70- mutants is enable to grow in GAL presence. All mutants present a reduction in specific activity but this defect in catalysis is not associated with reduction in substrate affinity. In time course experiments, performed in presence of DNA excess, the mutants present a more distributive mode of action. In the case of 434 mutants this loss of activity is associated with shift of cleavage/relegation equilibrium toward cleavage as confirmed also by cleavage assay. The mutants P431G present defect in relaxation caused by alteration in the control of strand rotation. Both with the Top1P431Gp and Top1P431G-70p mutant, a low, but reproducible level of DNA cleavage was observed in the absence of CPT . For the first the products of reaction are located in upper part of gel and corresponding to DNA substrates greater than 100 nucleotides in length. In the case of Top1P431G-70p mutant the products are placed in the lower part of gel and related to short DNA fragments. These results support a presence of interaction between N-terminal domain and hinge region. These results support a presence of interaction between N-terminal domain and hinge region. The N-terminal domain controls the substrate specificity, modulating the type and quality of protein-DNA interaction.
La DNA topoisomerasi I (Top1p) catalizza cambiamenti nella topologia del DNA mediante la formazione di un intermedio covalente enzima-DNA, che è reversibilmente stabilizzato dall’ agente antitumorale camptotecina (CPT).Studi cristallografici di una Top1p, mancante della porzione N-terminale, legata covalentemente ad un filamento di DNA, mostrano una proteina monomerica che lega il substrato come una tenaglia. In questa struttura sono individuabili un dominio core, che costituisce insieme al domino C-terminale la parte globulare della proteina e un dominio linker. La CPT lega l’ intermedio covalente limitando la flessibilità del dominio linker e consentendone la cristallizzazione. Precedentemente abbiamo dimostrato che la mutazione del residuo Ala653 in Pro determina un incremento nella tasso di riligazione rendendo il mutante Top1A653Pp resistente alla CPT (Fiorani, et al., 2003).Studi di dinamica molecolare suggeriscono che questa mutazione si associa ad un incremento nella flessibilità del dominio linker capace di alterare l’ equilibrio della reazione catalizzato dalla Top1p. Tuttavia i dati derivanti da studi strutturali, biochimici e di dinamica molecolare, condotti su questa proteina, forniscono scarse evidenze sul fatto che la flessibilità del dominio linker possa influenzare la geometria del sito attivo. Per verificare se l’ incremento nel tasso di riligazione causato dalla mutazione A653P, potesse sopprimere il difetto nella riligazione indotto dalla mutazione T718A, localizzata nel sito attivo, abbiamo realizzato un doppio mutante Top1 A652P-T718Ap (DM).Il doppio mutante è vitale, cataliticamente attivo sia in vivo che in vitro e resistente alla CPT. Questi risultati suggeriscono l’esistenza di interazioni a lungo raggio tra il dominio linker ed il sito attivo. L’ attività specifica del doppio mutante, sia in vivo che in vitro, dimostra la presenza di una riduzione dell’ affinità per il DNA, supportando l’ipotesi secondo cui alterazioni nella flessibilità o nell’orientamento di questo dominio influenzino profondamente la geometria del sito catalitico, modificando la cinetica del taglio e della riligazione catalizzata dalla Top1p. La struttura cristallografica del complesso ternario, realizzata in presenza dell’ analogo della CPT topotecano (TPT), suggerisce la presenza di due classi di mutazioni capaci di indurre resistenza all’ azione dell’ inibitore. La prima classe include i cambiamenti nei residui che interagiscono direttamente con il farmaco, mentre la seconda comprende i residui che, se mutati, determinano un alterazione nelle interazione stabilite con il DNA e la tasca di legame della CPT. La mutazione Thr729Ala si trova entro un cluster idrofobico situato nel dominio Cterminale e come tale, appartiene ad una terza classe di mutazioni capaci di conferire resistenza alla CPT senza interagire direttamente con il farmaco o il DNA. La mutazione Thr729Ala è stata identificata per la prima volta in linee cellulari tumorali PC-7/CPT resistenti al CPT-11( ironotecano un analogo della CPT) (Kubota et al., 1992). Tuttavia, i nostri dati rivelano che gli stessi effetti non sono evidenziabili se l’ enzima viene espresso nel lievito Saccaromyces cerevisiae. Se si osserva la struttura proteica il residuo Thr729 è distante dal sito di legame del farmaco per cui non è chiaro come una sua mutazione possa modificare la dinamica dell’ interazione tra CPT e complesso binario. La Thr729 si trova nel dominio C-terminale a 12.4 Å dalla tirosina catalitica e 13,1Å dal residuo Asn722 che stabilisce un legame mediato dall’ acqua con la CPT. La mutazione Asn722Ser, che determina un accorciamento della catena laterale, induce una condizione di resistenza (Fertala et al., 2000). Redinbo e coautori hanno ipotizzato che le basi della resistenza alla CPT, causata da mutazioni del residuo 729, siano dovute ad uno spostamento del residuo Asn722 con conseguente soppressione del legame con le CPTs (Chrencik, et al., 2004). per confermare questa ipotesi abbiamo analizzato il comportamento di altre tre sostituzioni: Lys, Pro e Glu. Queste sostituzioni sono state scelte per la loro carica positiva o negativa, nel caso della Lys e del Glu, e per la capacità di distorcere l’?-elica nel caso della Pro. I risultati dimostrano che il residuo Thr729 svolge un ruolo chiave nel mantenimento della corretta geometria della tasca idrofobica collocata nella regione C-terminale. Infatti, la mutazione Thr729Ala produce un’ enzima che presenta un attività in vivo, in vitro e una sensibilità alla CPT del tutto paragonabili all’ enzima WT. Quando la Thr729 è mutata in Lys o Glu si evidenzia una condizione di resistenza alla CPT ,che si traduce in una sensibilità alterata nel caso della mutazione Thr729Pro. Inoltre, il mutante Thr729Glu evidenzia forti difetti nell’ interazione con il substrato suggerendo che il mantenimento della corretta geometria della regione C-terminale è necessario per preservare le interazioni tra l’enzima ed il DNA durante la progressione del ciclo catalitico. Gli esperimenti di dinamica molecolare forniscono un‘interpretazione strutturale e dinamica del ruolo svolto dalla Thr729 nelle interazioni a lungo raggio presenti tra proteina e DNA (Chillemi et al. 2008). La catena laterale della Thr729 forma un legame idrogeno con il gruppo idrossilico della Tyr619, stabilizzando i contatti tra dominio C-terminale e la regione del subdominio III. La struttura più completa della Top1 contiene una porzione dell’ N-terminale che va dal residuo Ile 215 alla Gly201. Questi quindici aminoacidi sono impaccati vicino al subdominio I, al dominio C-terminale e alla regione hinge ove costituiscono un cluster idrofobico conservato in tutte le topo isomerasi IB eucaristiche (Redinbo et al., 2000). Questa porzione del dominio N-terminale interagisce con l’?-elica del perno ed, in particolare, il Trp 205 è vicino all’ Arg434, situata all’ inizio dell’ elica hinge. All’apice dell’hinge è presente un loop piegato contenete la Pro431. Per verificare se tale caratteristica strutturale è causata dalla presenza della prolina abbiamo mutato questo residuo in glicina, un aminoacido privo di catena laterale. In questo modo è possibile valutare se il ripiegamento del loop è causato dalla prolina e se tale conformazione possiede un significato funzionale. Per chiarire il ruolo delle interazioni presenti tra questa regione e la porzione N-terminale della proteina tutti i mutanti sono stati realizzati nella forma full lenght e Topo70. Inoltre il residuo Arg434 è stato mutato in Ala e Cys. La prima sostituzione è stata scelta per la sua capacità di alterare l’ intorno chimico sia strutturalmente che elettrostaticamente , la seconda per la capacità di formare un ponte disolfuro nel doppio mutante W205C-R434C, in cui la regione perno è ancorata covalentemente al domini N-terminale. Tutti i mutanti risultano letali quando espressi in lievito, la loro letalità non è dipendente dalla presenza del dominio N-terminale, poiché anche i mutanti Topo70 sono incapaci di formare colonie in condizioni di espressione. Tutte le proteine presentano una riduzione nell’ attività specifica, che tuttavia, non è associata ad un calo nell’ affinità per il substrato. Nelle cinetiche di rilassamento in eccesso di DNA, i mutanti presentano un comportamento distributivo. Nel caso dei mutanti del residuo 434 questa perdita di attività è associata ad uno spostamento dell’ equilibrio di reazione verso il taglio, come confermato dai saggi di taglio. I mutanti Top1P431Gp e Top1P431G70-p presentano difetti nel rilassamento del DNA probabilmente imputabili ad alterazioni nel controllo della rotazione del filamento a valle del sito di taglio. Nei saggi di taglio per entrambi è presente un basso ma riproducibile accumulo di complessi in assenza dell’ inibitore. Per il mutante Top1P431Gp i prodotti della reazione sono localizzati nella parte superiore del gel e corrispondenti a frammenti di DNA più lunghi di 100 coppie di basi. Nel caso del mutante Top1P431G70-p i prodotti di reazioni sono posizionati nella metà inferiore del gel e quindi equivalenti a frammenti di DNA corti. Questi risultati confermano la presenza di interazioni tra dominio N-terminale e regione hinge; inoltre suggeriscono che la regione N-terminale controlli la specificità del riconoscimento modulando la qualità e il tipo d’interazione tra enzima e DNA.

Cellular and biochemical studies support a role for all five human RecQ helicases in DNA replication, however their specific functions during this process are unclear. In my thesis, I investigated the in vivo association of the five human RecQ helicases with three well-characterized human replication origins. I showed that only RECQ1 and RECQ4 associate with replication origins in a cell cycle-regulated fashion in unperturbed cells, while other RecQ helicases interact with replication origins only under replication perturbed conditions. Under endogenous conditions, RECQ4 is recruited to origins at late G1 after ORC and MCM complex assembly, while RECQ1 and additional RECQ4 are loaded at origins at the onset of S phase when licensed origins begin firing. Both proteins are lost from origins after DNA replication initiation, indicating either disassembly or tracking with the newly formed replisome. Cell proliferation, DNA synthesis, nascent origin DNA synthesis and the frequency of origin firing are reduced after RECQ1 depletion, and to a greater extent after RECQ4 depletion. Depletion of RECQ1, though not RECQ4, also suppresses replication fork rates in otherwise unperturbed cells. Loading of PCNA during S phase is affected by RECQ1 depletion while the RECQ4 depleted cells show defect in RPA and PCNA loading during S phase of the cell cycle. These results indicate that RECQ1 and RECQ4 are integral components of the human replication complex, and play distinct roles in DNA replication initiation and replication fork progression in vivo.

The research activity, the results of which are the subject of doctoral dissertation, focused on the potentials of DNA barcoding, a genomic approach that exploits a short DNA sequence, a barcode, from a standardized region of the plastid genome, mitochondrial and chloroplast, as a universal and unique identification marker for animal and plant species. The main goal was to test a new accurate and automatable method for the genetic traceability of agri-food products, both of animal (fish, crustaceans and molluscs) and plant origin (bean and grapevine). First of all, we chose the specimens for the analysis: we selected pure lines of bean (Phaseolus vulgaris L.), clones of grapevine (Vitis vinifera L.) and samples of fish, crustaceans and molluscs purchased in famous GDO or local market in Chioggia e Sottomarina. Regarding the selection of seafood samples to analyze, we proceeded with the collection of the marine species most commonly involved in fraudulent substitutions. The experimental procedure adopted were the genomic DNA isolation from 37 specimens followed by the amplification of three target regions, cox1 (cytochrome oxydase subunit I), cob (apo-cytochrome b) and 16S-rDNA (ribosomal RNA small subunit) genes. Once obtained these data, we proceeded with a sequence similarity search using BOLD and GenBank as reference databases and each of the sequences as query. Overall, the phenetic approach proved to be an efficient tool to ensure the correct detection of seafood composition and thus to control the label information. In fact, for most of the samples it was possible to confirm the origin of the meat declared on the label, except in five situations where it was impossible to establish with no doubt the origin of the samples flagging them as likely falsification cases, voluntary or by accident. Cox1 gene proved to be a valid target for traceability aims, except in three genera, Thunnus, Macruronus and Gadus, where the identification was more problematic. Finally, even if GenBank database still remains the best web tool for forensic purposes, BOLD database proved to be enough rich to allow the correct recognition of almost all the specimens. Regarding plant DNA barcoding, the goal was to test DNA barcoding strategy as a tool to assess the distinctiveness of species and varieties of pure lines and clones. In the case of bean, we selected 54 pure lines of Phaseolus vulgaris species, 24 Italian pure lines, 18 Mesoamerican landraces and 12 Andean landraces, along with a few P. coccineus, P. lunatus and Vigna unguiculata accessions adopted as reference standards and out-types. These samples were characterized by means of the amplification of 7 chloroplast and two nuclear regions followed by the application of a phenetic approach. The procedure confirmed to be a powerful technique to correctly separate different species, whereas at the varietal level it revealed to be scarcely informative to discriminate gene pools and to identify varieties within P. vulgaris. Thus a second approach, the character-based system, was tested and it allowed to detect within P. vulgaris species a total of 16 haplotypes corresponding to as many subgroups, each one made up by Mesoamerican or Andean accessions along with Italian accessions that clustered with one or the other gene pool. Finally, a third case study is represented by V. vinifera and the potentials of DNA barcoding approach to distinguish grapevine cultivars used in the production of wines. We proceeded with the selection of 123 grapevine cultivars along with other 5 species of Vitis (V. rupestris, V. riparia, V. labrusca, V. cinerea e V. berlandieri) adopted as reference standards and out-types. After a preliminary analysis of the chloroplast DNA that resulted to be monomorphic, we decided to shift to the nuclear genome amplifying four ESTs and the GAI1 (gibberellins insensitive-like) gene. The analysis is still ongoing, but the preliminary results lead to think that a few haplotypes exist within V. vinifera and they could be use to resolve frequent cases of synonymies and homonymies in grapevine. Furthermore, an economically valuable application may be the exploitation of these haplotypes cultivar-specific for the genetic traceability of wines to avoid cases of falsification.
L’attività di ricerca, i cui risultati sono oggetto della dissertazione di dottorato, ha riguardato lo studio delle potenzialità applicative del DNA barcoding, una tecnica molecolare volta all’identificazione degli organismi sulla base dei polimorfismi di specifiche sequenze nucleotidiche localizzate nei genomi plastidiale, mitocondriale e cloroplastico. Il progetto di ricerca ha previsto l’impiego di questo approccio per il riconoscimento di specie ai fini della tracciabilità genetico-molecolare di prodotti agro-alimentari, sia di origine animale (pesci, molluschi e crostacei) che vegetale (fagiolo e vite). Inizialmente si è proceduto all’individuazione degli organismi su cui condurre l’analisi: si sono collezionate linee pure di fagiolo (Phaseolus vulgaris L.), cloni di vite (Vitis vinifera L.) e campioni di pesci, crostacei e molluschi acquistati presso famose GDO o ai mercati locali di Chioggia e Sottomarina. In particolare, per quanto concerne la scelta delle specie ittiche su cui condurre l’analisi, si è svolta un’estesa indagine di mercato con l’intento di individuare le specie maggiormente coinvolte in falsificazioni alimentari, cioè sostituzione di specie pregiate con altre di valore inferiore. Si è successivamente proceduto alla purificazione di 37 campioni di DNA genomico e alla loro caratterizzazione dal punto di vista molecolare mediante amplificazione e sequenziamento di specifici geni mitocondriali, quali cox1 (Cytochrome oxydase subunit I), 16S-rDNA (16S small ribosomal subunit RNA) e cob (cytochrome b). Una volta acquisiti questi dati, l’interrogazione di due banche dati disponibili on line, BOLD per il gene cox1 e GenBank per tutti e tre i geni, ha consentito di identificare l’origine dei campioni confermando nella maggioranza dei casi quanto dichiarato nell’etichetta di accompagnamento del prodotto alimentare. In cinque situazioni non è stato possibile stabilire con certezza l’origine del campione e questo potrebbe indicare possibili casi di sostituzione, fraudolenta o accidentale. Il DNA barcoding pertanto è risultato utile ai fini dell’identificazione di specie in tutti e tre i taxa studiati, pesci, molluschi e crostacei, e il gene cox1 si è dimostrato un ottimo target per questi scopi eccetto che in tre casi particolari, i generi Thunnus, Macruronus e Gadus. Inoltre è risultato evidente che nonostante GenBank persista come la banca dati più ricca in termini di numero di sequenze depositate, il BOLD sta rapidamente incrementando la quantità di informazioni contenute al suo interno lasciando presupporre che in breve tempo diventerà la banca dati di riferimento per studi di genetica forense e di tracciabilità genetica. Per quanto riguarda le specie vegetali, l’obiettivo era l’identificazione univoca di specie, e soprattutto delle loro varietà quando fondate su un solo genotipo (linee pure, ibridi e cloni). Nel caso di fagiolo, si sono isolati i DNA genomici da 54 varietà di Phaseolus vulgaris, 18 provenienti dal Centro America, 12 dal Sud America e 24 line pure coltivate e commercializzate in Italia, insieme con alti 6 campioni usati come fuori gruppo (Phaseolus coccineus, Phaseolus lunatus e Vigna unguiculata). Sono risultate indispensabili indagini preliminari di polimorfismi di singoli geni al fine di determinare la variabilità genetica tra le varietà e la tracciabilità genetica di singole varietà. La caratterizzazione, tramite l’amplificazione di 7 differenti regioni cloroplastiche e due nucleari seguita da un approccio fenetico, ha confermato le potenzialità della tecnica come strumento efficace per la distinzione delle specie, mentre è risultata scarsamente informativa per il riconoscimento di singole varietà. Da qui si è rivelata necessaria l’adozione di un approccio alternativo, basato sulla determinazione della composizione nucleotidica e del polimorfismo a carico di ciascun gene esaminato, che ha permesso di definire 16 aplotipi corrispondenti ad altrettanti sottogruppi varietali, ciascuno costituito da accessioni Mesoamericane o Andine insieme con le varietà Italiane. Infine l’applicazione del DNA barcoding per la distinzione di cultivar di vite ha richiesto l’abbandono dello studio del genoma cloroplastico, troppo poco variabile, a favore di quello nucleare. Si sono isolati i DNA genomici da 123 cultivar di Vitis vinifera e da altre 5 specie (V. rupestris, V. riparia, V. labrusca, V. cinerea e V. berlandieri) e si sono amplificati 4 EST ed il gene GAI1 (gibberellins insensitive-like). L’analisi bioinformatica è ancora in corso, ma risultati preliminari fanno ipotizzare l’esistenza di aplotipi cultivar-specifici che potrebbero venir impiegati in futuro per risolvere i frequenti casi di sinonimie ed omonimie diffusi all’interno di questa specie. Infine un’altra interessante applicazione da un punto di vista economico potrebbe essere l’impiego di questi aplotipi cultivar-specifici per la tracciabilità genetica dei vini e la tutela delle denominazioni controllate da casi di falsificazione e concorrenza sleale.

In metazoan cells the DNA replication origins are not well defined. Differently from what observed for bacteria cells and for budding yeast, in metazoan the origins does not show a conserved sequence and they appear to be specified by many factors. In order to better understand the mechanisms involved in the origin specification, many studies have been done to identify the proteins involved in the recognition and activation of the origins. From these kind of analysis is emerging that, beside the wellknown proteins of the pre replicative complex, also other factors might be involved. Between these, the HOX proteins seem to be able to play a role in the origin activity. One of the first studies of this involvement was done by our group and leads to the identification of three homeotic proteins able to specifically bind in vitro the human lamin B2 origin. Thus, in the study conducted during this PhD program, was investigated the involvement of one of these homeotic proteins, namely HOXC13, with human DNA replication origins and with replicative complexes. We found an interaction of HOXC13 with two crucial factors of the pre Replication Complex (pre-RC), ORC1 and Cdc6 and that HOXC13 binds a good fraction of the origins, in particular the early replicating ones, like the lamin B2 origin and other known human origins. The HOXC13 protein is bound to origin chromatin, at least for the lamin B2 origin, at a precise site within the pre-RC at specific moments of the cell cycle. Interaction with the origin occurs within the area protected by the pre-RC in G1, very close to the start sites of leading strand synthesis and to the binding sites of ORC1, ORC2, Cdc6, topoisomerase (topo) I and topo II. The protein is absent from the origin in M and appears on it at the beginning of G1, reach a peak at G1/S and as synthesis starts, the interaction of HOXC13 with the origin fades, in parallel with the transition from this large pre-RC to a smaller and differently organized post-RC. Recently also other HOX proteins have been identify as proteins involved in regulation processes of DNA replication, suggesting that the interaction of HOXC13 with the origins might occur in a multi-homeotic proteins complex. Depletion of one of these proteins however is compatible with the continuation of the cell cycle and, according with what observed for the other homeotic proteins, we found that also the depletion of HOXC13 does not alter cell cycle progression or S phase entry. This is probably due to the redundancy of homeotic proteins and indicates a relatively generic function for the HOX proteins. Among the identified elements influencing the choice and the activity of a sequence as DNA replication origin, much relevance is assumed by the chromatin structure and topology of DNA. Therefore, we analysed the effects of chromatin structure disruption using Tricostatin A, a histone deacetylase inhibitor. The alteration of chromatin caused by this treatment not only sharply reduces origin function, but also disturbs the binding of replication complex members like HOXC13 and the well known Cdc6 to the DNA replication origins, while does not affect the binding of other unrelated proteins like USF1. On the basis of this finding, we infer that an appropriate chromatin organization and DNA topology strongly influence the binding between factors of the pre Replication Complex and DNA replication origins. This influence could be a key element in origin specification. The described interactions are not restricted to a single origin nor to a single homeotic protein, leading us to conclude that HOX proteins, probably in the context of a multi-protein homeotic effectors, contribute to recruit and stabilize the replicative complexes onto early replicating origins, in presence of specific chromatin and topological configurations. The relevance of HOXC13 in DNA replication is also underlined by its involvement in oncogenesis, clearly demonstrated in acute myeloid leukaemia when HOXC13 is fused with NUP98 protein.

Genome maintenance and stability are essential goals for all the organisms in order to transfer the correct genetic information to the progeny and to keep fully functional the cellular metabolism. In eukaryotic cells, the presence of DNA lesions causes the activation of an evolutionary conserved mechanism called the DNA damage checkpoint that arrests the cell cycle and stimulates the repair pathways. Double strand breaks (DSBs) are deleterious lesions that can be a serious threat for the cell. In fact, the formation of only one DSB is enough to activate a robust checkpoint response. This DNA lesion is processed by several factors leading to the checkpoint factors recruitment and to the homologous recombination repair. After lesion repair the checkpoint is switched off through a process called recovery; however it has been demonstrated that damaged cells are able to inactivate the checkpoint and restart the cell cycle also in the presence of a persistent DNA lesion, through a checkpoint adaptation process. The reason why this process occurs is not understood, but it has been related to the unrestrained proliferation of cancer cell. In my laboratory we are interested in shedding light on the molecular mechanism of these checkpoint inactivation processes and in the characterization of the involved factors. During the PhD I focused on the characterization of the functions and regulation of some factors already known to play a role in DSB ends processing and checkpoint switch off: the polo kinase Cdc5, the DNA translocase Tid1/Rdh54 and the nuclease-associated protein Sae2. First of all we found that high levels of Cdc5 lead to checkpoint switch off and cell cycle re-enter. Relying on this data we decide to perform a biochemical screening in order to identify the Cdc5 targets in presence of DNA damage. This biochemical screening was based on a GST pulldown approach, coupled with tandem mass spectrometry protein identification. As expected, we identify many interactors and among them we found the repair protein Sae2. Interestingly, we found that in presence of elevated levels of Cdc5, Sae2 is hyperphospholylated and binds strongly to the DSB ends. In order to understand the functional role of the Cdc5-Sae2 interaction, I mutagenized different putative Cdc5 binding sites in Sae2. It turned out that Cdc5 binds a C-terminal region of Sae2, which is conserved in other eukaryotes orthologs. The obtained Sae2 mutants give us interesting results that can be useful for the proper comprehension of the Sae2 function in DNA damage response. Indeed in this thesis I will present preliminary results on the characterization of the Sae2 role in the recovery process. I was also involved in a project with the aim to study the regulation of Tid1/Rdh54 in the presence of DSB. Tid1 belongs to the Swi2/Snf2 family of chromatin remodellers, is an ATP-dependent DNA translocase able to induce DNA structure remodelling, Rad51 removal from double strand DNA and promote D-loop formation during homologous recombination. Moreover this protein has also a puzzling function in checkpoint inactivation during adaptation since TID1 deletion causes a permanent G2/M block in the presence of one irreparable DSB. I found that Mec1 and Rad53 checkpoint kinases, through a process that requires also the recombination factor Rad51, phosphorylate Tid1 in the presence of DSBs. I also found that Tid1 is recruited on to the DSB site, and that its ATPase activity is dispensable both for the loading and the phosphorylation of the protein. We believe that Tid1 phosphorylation is important to stabilize the binding of the protein on the lesion and to regulate its functional role during checkpoint adaptation.

Maize (Zea mays) is an important source of proteins for human and animal nutrition. However, because of the lack of lysine and the low content in methionine and tryptophan, maize’s proteins are of low quality. These deficiencies mainly result from the low levels of these essential amino acids in the zein storage proteins, which account for 50% of the total protein in mature seed. In this context, the first aim of this PhD thesis has been to develop artificial zein genes encoding for polypeptides with a higher content in lysine and methionine, and capable to be sorted and correctly accumulated into the endosperm, as occur for natural zein polypeptides. Two strategies have been employed for maize bio-fortification. First, we exploited the natural heterogeneity among α-zein genes to create a synthetic gene, ZRK, in which six arginine residues have been substituted with lysine. Then, by combining the N-terminal methionine-rich G3 sequence and the C-terminal lysine-rich region of Histone3 and Histone4 of maize, the G3H3 and G3H4 artificial genes were created, respectively. In vitro and in vivo expression analyses of these genes showed that all synthetic proteins are synthesized and accumulated into the ER membranes of either the rabbit reticulocyte/canine membrane system or of transformed tobacco protoplasts. The second aim of this thesis has been to use the wide heterogeneity of zein gene family to obtain an intra-species recognition tool, or individual barcode, for inbreds and Lombard varieties discrimination. Lombard varieties and maize inbreds were analysed by 2D gel protein fractionations and DNA gel blot analyses. For each genotype the 2D and Southern blot pattern were converted into a binary code, and then into a barcode. In both the approaches, each genotype was univocally identified making zeins a valuable tool for identification of maize germplasm.