Gotowa bibliografia na temat „DNA virus- G-quadruplex secondary structures”

G-quadruplexes (G4s) are noncanonical secondary nucleic acid structures constituted by stacking of guanine rich planar shaped tetrad formations that form a complex. G4s are implicated for various important roles in key cellular processes transcription, translation, telomere maintenance, epigenetic regulation, replication, and recombination. G-quadruplexes were first discovered as important structures in oncology, but for the past decade its relevance in viruses is becoming more evident. Human herpesviruses are DNA viruses of the Herpesviridae family and are unique in characteristic with two types of infection which can be distinguished by lytic and latency establishment in the host. During latency the virus maintains lifelong dormancy and intermittently undergoes reactivation, causing the host medical problems. Recently there are increasing number of reports regarding role of G4s in viral genomes and the potential antiviral efficacy of G4 ligands, including G4s in latency. Many results suggest viral G4s play significant roles in the virus life cycle and treatment of G4 ligands exhibit antiviral activities in both lytic and latent infections. In this review, the importance of G4s in herpesvirus genomes will be introduced with the potent G4 ligands used to study these mechanisms and finally explain the distinct functional properties of each G4 ligands.

The herpes simplex virus 1 (HSV-1) genome is extremely rich in guanine tracts that fold into G-quadruplexes (G4s), nucleic acid secondary structures implicated in key biological functions. Viral G4s were visualized in HSV-1 infected cells, with massive virus cycle-dependent G4-formation peaking during viral DNA replication. Small molecules that specifically interact with G4s have been shown to inhibit HSV-1 DNA replication. We here investigated the antiviral activity of TMPyP4, a porphyrin known to interact with G4s. The analogue TMPyP2, with lower G4 affinity, was used as control. We showed by biophysical analysis that TMPyP4 interacts with HSV-1 G4s, and inhibits polymerase progression in vitro; in infected cells, it displayed good antiviral activity which, however, was independent of inhibition of virus DNA replication or entry. At low TMPyP4 concentration, the virus released by the cells was almost null, while inside the cell virus amounts were at control levels. TEM analysis showed that virus particles were trapped inside cytoplasmatic vesicles, which could not be ascribed to autophagy, as proven by RT-qPCR, western blot, and immunofluorescence analysis. Our data indicate a unique mechanism of action of TMPyP4 against HSV-1, and suggest the unprecedented involvement of currently unknown G4s in viral or antiviral cellular defense pathways.

Nucleosomes were reconstituted in vitro from a fragment of DNA spanning the simian virus 40 minimal replication origin. The fragment contains a 27-base-pair palindrome (perfect inverted repeat). DNA molecules with stable cruciform structures were generated by heteroduplexing this DNA fragment with mutants altered within the palindromic sequence (C. Nobile and R. G. Martin, Int. Virol., in press). Analyses of the structural features of the reconstituted nucleosomes by the DNase I footprint technique revealed two alternative DNA-histone arrangements, each one accurately phased with respect to the uniquely labeled DNA ends. As linear double-stranded DNA, a unique core particle was formed in which the histones strongly protected the regions to both sides of the palindrome. The cruciform structure seemed to be unable to associate with core histones and, therefore, an alternative phasing of the histone octamer along the DNA resulted. Thus, nucleosome positioning along a specific DNA sequence appears to be influenced in vitro by the secondary structure (linear or cruciform) of the 27-base-pair palindrome. The formation of cruciform structures in vivo, if they occur, might therefore represent a molecular mechanism by which nucleosomes are phased.

Nucleosomes were reconstituted in vitro from a fragment of DNA spanning the simian virus 40 minimal replication origin. The fragment contains a 27-base-pair palindrome (perfect inverted repeat). DNA molecules with stable cruciform structures were generated by heteroduplexing this DNA fragment with mutants altered within the palindromic sequence (C. Nobile and R. G. Martin, Int. Virol., in press). Analyses of the structural features of the reconstituted nucleosomes by the DNase I footprint technique revealed two alternative DNA-histone arrangements, each one accurately phased with respect to the uniquely labeled DNA ends. As linear double-stranded DNA, a unique core particle was formed in which the histones strongly protected the regions to both sides of the palindrome. The cruciform structure seemed to be unable to associate with core histones and, therefore, an alternative phasing of the histone octamer along the DNA resulted. Thus, nucleosome positioning along a specific DNA sequence appears to be influenced in vitro by the secondary structure (linear or cruciform) of the 27-base-pair palindrome. The formation of cruciform structures in vivo, if they occur, might therefore represent a molecular mechanism by which nucleosomes are phased.

Abstract The human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3, A3) family member proteins can deaminate cytosines in single-strand (ss) DNA, which restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons, and other viruses such as hepatitis B virus, but can cause a mutator phenotype in many cancers. While structural information exists for several A3 proteins, the precise details regarding deamination target selection are not fully understood. Here, we report the first parallel, comparative analysis of site selection of A3 deamination using six of the seven purified A3 member enzymes, oligonucleotides having 5′TC3′ or 5′CT3′ dinucleotide target sites, and different flanking bases within diverse DNA secondary structures. A3A, A3F and A3H were observed to have strong preferences toward the TC target flanked by A or T, while all examined A3 proteins did not show a preference for a TC target flanked by a G. We observed that the TC target was strongly preferred in ssDNA regions rather than dsDNA, loop or bulge regions, with flanking bases influencing the degree of preference. CT was also shown to be a potential deamination target. Taken together, our observations provide new insights into A3 enzyme target site selection and how A3 mutagenesis impacts mutation rates.

ABSTRACT Homologs of the small tegument protein encoded by the UL11 gene of herpes simplex virus type 1 are conserved throughout all herpesvirus subfamilies. However, their function during viral replication has not yet been conclusively shown. Using a monospecific antiserum and an appropriate viral deletion and rescue mutant, we identified and functionally characterized the UL11 protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL11 encodes a protein with an apparent molecular mass of 10 to 13 kDa that is primarily detected at cytoplasmic membranes during viral replication. In the absence of the UL11 protein, viral titers were decreased approximately 10-fold and plaque sizes were reduced by 60% compared to wild-type virus. Intranuclear capsid maturation and nuclear egress resulting in translocation of DNA-containing capsids into the cytoplasm were not detectably affected. However, in the absence of the UL11 protein, intracytoplasmic membranes were distorted. Moreover, in PrV-ΔUL11-infected cells, capsids accumulated in the cytoplasm and were often found associated with tegument in aggregated structures such as had previously been demonstrated in cells infected with a PrV triple-mutant virus lacking glycoproteins E, I, and M (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Thus, the PrV UL11 protein, like glycoproteins E, I, and M, appears to be involved in secondary envelopment.

A cursory look at any textbook image of DNA replication might suggest that the complex machine that is the replisome runs smoothly along the chromosomal DNA. However, many DNA sequences can adopt non-B form secondary structures and these have the potential to impede progression of the replisome. A picture is emerging in which the maintenance of processive DNA replication requires the action of a significant number of additional proteins beyond the core replisome to resolve secondary structures in the DNA template. By ensuring that DNA synthesis remains closely coupled to DNA unwinding by the replicative helicase, these factors prevent impediments to the replisome from causing genetic and epigenetic instability. This review considers the circumstances in which DNA forms secondary structures, the potential responses of the eukaryotic replisome to these impediments in the light of recent advances in our understanding of its structure and operation and the mechanisms cells deploy to remove secondary structure from the DNA. To illustrate the principles involved, we focus on one of the best understood DNA secondary structures, G quadruplexes (G4s), and on the helicases that promote their resolution.

The most common form of DNA is a right-handed helix or the B-form DNA. DNA can also adopt a variety of alternative conformations, non-B-form DNA secondary structures, including the DNA G-quadruplex (DNA-G4). Furthermore, besides stem-loops that yield A-form double-stranded RNA, non-canonical RNA G-quadruplex (RNA-G4) secondary structures are also observed. Recent bioinformatics analysis of the whole-genome and transcriptome obtained using G-quadruplex–specific antibodies and ligands, revealed genomic positions of G-quadruplexes. In addition, accumulating evidence pointed to the existence of these structures under physiologically- and pathologically-relevant conditions, with functional roles in vivo. In this review, we focused on DNA-G4 and RNA-G4, which may have important roles in neuronal function, and reveal mechanisms underlying neurological disorders related to synaptic dysfunction. In addition, we mention the potential of G-quadruplexes as therapeutic targets for neurological diseases.

Genomic complexity in non-Hodgkin’s Diffuse Large B-cell Lymphoma (DLBCL) leads to a treatment failure in ~40% of patients. Activation-Induced Cytosine Deaminase (AID), one of the enzymes involved in generating antibody diversity via class switching recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes in activated B-cells is one mechanism for the introduction of genomic lesions. In previous studies, AID was shown to preferentially bind to super-enhancer (SE) regions within the genome, but 26% of AID targets were not within the SE regions. The mechanism by which AID interacts with SE elements and its off-target interactions still remains a mystery. Recent evidence suggests that AID may cause genomic lesions in DLBCL via interaction with oncogenes such as MYC and BCL2 resulting in mutations and translocations. Sequences within the MYC promoter contain the four-nucleotide AID target sequence (WRCY) and highly G-rich sequences known to form G-quadruplex DNA secondary structures. We hypothesize that key DNA secondary structures act as recruiting elements for aberrant AID activity at promoters and SEs of key genes involved in the development of DLBCL. Here, we first sought to determine whether known AID DNA targets have the potential to form G-quadruplex DNA secondary structures. The data collected from activated mouse B-cells showed 90% of the AID targets contained sequences that could potentially form G-quadruplexes and the data collected from the human Ramos cell line showed 100% of the sequences had the potential to form G-quadruplexes. To further study our hypothesis we used the techniques circular dichroism (CD) and the electrophoresis motility shift assay (EMSA) to explore the potential interaction between AID and the BCL2 and MYC G-quadruplexes. We observed no significant interactions between AID and these two G-quadruplexes, however further experimentation with different conditions and molecular techniques may show interaction. Additional studies will not only provide key insight into the genomic instability within DLBCL, but will also provide a potential mechanism by which AID is recruited to its DNA targets.

The present manuscript contains a report of the research activity conducted for the Ph.D. project regarding the study of DNA G-quadruplex structures in a rare type of human cancer (Liposarcoma) and in the genome of two world-wide spread viruses: HIV-1 and HSV-1.
Il manuscritto contiene una descrizione e discussione dei risultati ottenuti in ambito del progetto di ricerca di dottorato riguardante lo studio delle strutture G-quadruplex delDNA in un tumore umani raro (il liposarcoma) e nel genoma di due virus a diffusione mondiale, quali: HIV-1 e HSV-1.

The folding of DNA molecule into non-canonical secondary structures has been shown to be implicated in many important biological processes which regulate cell proliferation and proteins expression. In particular one of these peculiar secondary structures, called G-quadruplex (G4), has been shown to potentially impair cancer development. G4 occurs along DNA sequences rich of consecutive guanines which can fold through Hoostein pairs by forming stacked planes of guanines tetrads. This conformation prevalently forms along the termini of chromosomes (telomeres) but also along the promoter sites of several oncogenes directly involved in many cancers. The G4 formation leads to an hindrance on DNA molecule which hinder the telomere elongation and transcription process. The result is a switching off of these mechanisms which are directly involved in cancer progression. Several factors can influence the G4 equilibria for example, saline conditions, temperature, pH, the binding with specific proteins as well as the presence of dehydrating cosolutes. Additionally, the overall structural feature of the G4 is strictly dependent upon the DNA sequence. As a results, different G4 can be identified inside the cells. In this project, we focused on the conformational study of the promotorial regions of EGFR and BRAF oncogenes since, on these sites the existence of G4 putative forming regions was found. In particular, the sequences at positions -272, -37 of EGFR and -176 of BRAF from the transcription start site were analyzed. Indeed, no previous literature data were reported about the structural equilibria in solution of these sequences. We found that our tested sequences are actually able to fold into G4 by setting the most proper experimental conditions and also close to the intracellular physiological environment (KCl 150 mM, pH 7.5). However, oncogenes are double stranded sequences and the folding of the complementary cytosine rich strand into i-motif (iM) can be involved in the switching off of gene transcription. Although, so far, no physiological evidence has been observed for i-motif conformation, here, we aimed to investigate also the cytosine rich strand conformation, to assess if this folding in the case of our sequences is compatible with the physiological conditions and if it can synergically works with the G4 to destabilize the double strand. Our data showed that in physiological condition the preferential form is represented by the double strand . However, some selected ligands showed to shift the DNA B-form toward the non canonical conformation. Indeed, here we implemented our work with the screening of two libraries of compounds in order to find a selective and efficient binder. We carried on the binding study of anthraquinones and naphthalene diimides derivatives, known to have the chemical features of efficient G4 binders. These ligands were first tested on different G4 templates, known to be validated models for G4 binding study, and their efficiency on G4 has been compared with the double strand. The most G4 selective derivatives were than investigated towards our oncogenic G4s. Although more work is required to identify a lead compound, we were able to demonstrate how the use of asymmetrical substitution pattern on a aromatic core can implement the selectivity among different G4s. Finally, in order to map the occurrence of G4 conformation in vivo, we set up a novel technique which consists in an in vivo footprinting protocol. This work, performed at University of Mississippi, Oxford, MS (USA), under the supervision of Dr Tracy A. Brooks, should provide novel insight on the G4 formation in the cells according to their physiological and environmental conditions
Molti studi dimostrano che l’assunzione di strutture “non canoniche” da parte della molecola di DNA sia coinvolto in molti importanti processi biologici che regolano la proliferazione cellulare e l’espressione proteica. In particolare, è stata dimostrata l’implicazione di una di queste particolari strutture secondarie, chiamata G-quadruplex (G4), nel blocco della progressione del cancro. La struttura G4 è propria di sequenze di DNA ricche in guanine consecutive che assemblandosi tramite legami di Hoostein, formano piani di tetradi di guanine impilati tra loro. Questa particolare conformazione si forma prevalentemente lungo i tratti terminali dei cromosomi, i telomeri, ma anche lungo siti promotoriali di diversi oncogeni coinvolti in molti tipi di cancro. La formazione del G4 porta ad una sorta di ingombro sulla molecola di DNA che inibisce l’elongazione del telomero e i processi di trascrizione. Questo porta ad uno “spegnimento” di questi meccanismi che sono direttamente coinvolti nello sviluppo del cancro. Molti fattori possono influenzare gli equilibri delle conformazioni G4, per esempio, le condizioni saline, la temperatura, il pH, il legame con specifiche proteine, così come la presenza di cosoluti. Inoltre, la struttura globale del G4 é rigorosamente dipendente dalla sequenza oligonucleotidica. Pertanto, diverse strutture G4 possono essere identificate a livello cellulare. In questo progetto, è stato condotto uno studio conformazionale di regioni promotoriali degli oncogeni EGFR e BRAF, dal momento che, su questi oncogeni è stata riscontrata la presenza di regioni “G-rich” (ricche in guanine) potenzialmente in grado di assumere una struttura G4. In particolare, sono state analizzate le sequenze a partire dalle posizioni -272, -37 di EGFR e -176 di BRAF dal “transcription start site” (sito di inizio della trascrizione). Finora, non sono presenti dati in letteratura riguardanti la caratterizzazione strutturale di queste sequenze in soluzione. Con questo studio, è stata dimostrata la capacità delle suddette sequenze di assumere una conformazione G4 nelle idonee condizioni sperimentali e soprattutto in un ambiente che mimi quello fisiologico (150mM KCl e pH 7.5). Poiché gli oncogeni sono sequenze a doppio filamento, anche la conformazione i-motif assunta dal filamento complementare ricco in citosine (“C-rich”) può essere coinvolta nella regolazione del processo di trascrizione genica. Tuttavia, sinora non è stata riscontrata alcuna rilevanza fisiologica della conformazione i-motif. In questo lavoro, è stata caratterizzata anche la conformazione assunta dal filamento “C-rich”, in particolare se essa possa esistere in condizioni fisiologiche e se fosse in grado di destabilizzare la doppia elica insieme al G4. I dati ottenuti dimostrano che in condizioni fisiologiche la forma prevalente è il doppio filamento. Tuttavia, è stato dimostrato come alcuni ligandi siano in grado di spostare l’equilibrio del DNA dalla sua forma di doppia elica-B, verso le conformazioni non canoniche. È stato infatti condotto uno studio su due librerie di composti con lo scopo di evidenziare un composto selettivo ed efficace. Ci siamo focalizzati su derivati antrachinonici e di naftalendiimidi noti come efficaci ligandi per il G4. Questi composti sono stati prima testati su diversi templati G4, noti per essere dei modelli validati per lo studio di binding sul G4. Quindi la loro efficienza sul G4 è stata poi comparata a quella sul doppio filamento. I derivati più selettivi verso il G4 sono stati poi testati su G4 oncogenici. Sebbene una continuazione dello studio fosse necessaria per identificare un composto “lead”, con questo lavoro è stato dimostrato come l’uso di una sostituzione asimmetrica sull’anello aromatico possa implementare la selettività tra più G4. Infine, per identificare la formazione del G4 in vivo, è stata messa a punto una nuova tecnica che consiste in un protocollo di footprinting in vivo. Questo lavoro, svolto nell’Università del Mississippi, Oxford, MS (USA) sotto la supervisione della dr.ssa Tracy A. Brooks, dovrebbe fornire nuovi sviluppi per la formazione del G4 nelle cellule in accordo con le loro condizioni fisiologiche

In this dissertation, the formation of unusual G-quadruplexes in the critical regions of the c-myb and hTERT promoters for control of promoter activity was investigated.The c-myb promoter contains three copies of an almost perfect (GGA)4 sequence. We demonstrate that the each (GGA)4 repeat forms a tetrad:heptad G-quadruplex and any two of the three can intramolecularly dimerize to form T:H:H:T G-quadruplexes. The three T:H:H:T G-quadruplex combinations are of differing degrees of stability and can be further stabilized by G-quadruplex interactive compounds. We also demonstrate that the c-myb G-quadruplex forming region is a critical transcriptional regulatory element and interacts with various nuclear proteins including MAZ (Myc Associated Zinc finger protein). The data from luciferase reporter assay show that the c-myb GGA repeat region plays dual roles as a transcriptional activator and an inhibitor by serving as binding sites for the activators and by forming G-quadruplex structures in the region, respectively. Furthermore, we show that MAZ is a transcriptional repressor of the c-myb promoter and binds to both the double-stranded and T:H:H:T G-quadruplex-folded conformations of the GGA repeat region of the c-myb promoter.The hTERT core promoter contains a G-rich region of 12 consecutive G-tracts, which includes three critical Sp1 binding sites. Although this G-rich region has the potential to form multiple G-quadruplexes, our investigation on the full-length G-rich sequence demonstrate that the G-rich region forms a unique G-quadruplex structure in which two tandem intramolecular G-quadruplex structures are present, consisted of one G-quadruplex formed by the G-tracts 1-4 and the other formed by the G-tracts 5, 6, 11, and 12. We also demonstrate that the latter unusual structure contains a 26-base middle loop that likely forms a hairpin structure and is more stable than the other conventional G-quadruplex. Significantly, the formation of this unusual tandem G-quadruplex structure in the full-length will disable all three critical Sp1 binding sites, which will dramatically downregulate hTERT expression. G-quadruplex formation in the hTERT promoter suggests that the effect of G-quadruplex interactive ligands on telomerase inhibition and telomere shortening may be exerted by the direct interaction between the hTERT G-quadruplex structure and the ligands.

In this dissertation, we explore the transcriptional regulatory roles of Gquadruplex- forming motifs and the involvement of specific transcriptional factors, which interact with the same elements, in the control of human c-myc and bcl-2 gene expression. The G-quadruplex structures within the NHE III1 region of the c-myc promoter and their ability to repress transcription has been well established. However, a longstanding unanswered question is how these stable DNA secondary structures are transformed to activate c-myc transcription. NDPK-B has been recognized as an activator of c-myc transcription via interactions with NHE III1 region of the c-myc gene promoter. Through the use of RNAi, we confirmed the transcriptional regulatory role of NDPK-B. We demonstrate that NDPK-B has DNA binding activity and the nuclease activity results from a contaminating protein. NDPK-B preferentially binds to the singlestranded guanine-rich strand of the c-myc NHE III₁. Potassium ions and G-quadruplexinteractive agents, which stabilize G-quadruplex structures, had an inhibitory effect on NDPK-B DNA binding activity. Based on our studies, we have proposed a stepwise trapping-out of the NHE III1 region in a single-stranded form, thus allowing singlestranded transcription factors to bind and activate c-myc transcription. This model provides a rationale for how the stabilization of G-quadruplexes within the c-myc gene promoter region can inhibit NDPK-B from activating c-myc transcription. Similarly, the human bcl-2 gene contains a GC-rich region within its promoter region, which is critical in the regulation of bcl-2 expression. We demonstrate that the guanine rich strand within this region can form three intramolecular G-quadruplex structures. Based on NMR studies, the central G-quadruplex forms a mixed parallel/antiparallel structure with three tetrads connected by loops of one, seven, and three bases. The Gquadruplex structures in the bcl-2 promoter extends beyond the ability to form any one of three separate G-quadruplexes to each having the capacity to form either three or six different loop isomers. This suggests that targeting these individual structures could lead to different biological outcomes. We also found that Telomestatin upregulates bcl-2 gene expression, which we propose is a result of inhibiting the binding of the WT1 repressor protein by the formation of a drug-stabilized G-quadruplex structure.

Angiogenesis is known to be induced and maintained in tumors by the constant expression of the hypoxia inducible factor 1 alpha (HIF-1α) and human vascular endothelial growth factor (VEGF). In fact, tumor recurrence, aggressive metastatic legions and patient mortality rates are known to be positively correlated with overexpression of these two proteins. The HIF-1α and VEGF promoters contain a polypurine/polypyrimidine (pPu/pPy) tract, which are known to play critical roles in their transcriptional regulation, and are structurally dynamic where they can undergo a conformational transition between B-DNA, single stranded DNA and atypical secondary DNA structures such as G-quadruplexes and i-motifs. We hypothesize that the i-motif and G-quadruplex structures can form within the pPu/pPy tracts of the HIF-1α and VEGF proximal promoters, which play important roles in the transcriptional regulation of these genes by acting as scaffolds for alternative transcription factor binding sites. The purpose of this dissertation was to elucidate the transcriptional regulation of the HIF-1α and VEGF genes through the atypical DNA structures that form within the pPu/pPy tracts of their proximal promoters. We investigated the interaction of the C-rich and guanine-rich (G-rich) strands of both of these tracts with transcription factors heterogeneous nuclear ribonucleoprotein (hnRNP) K and nucleolin, respectively, both in vitro and in vivo and their potential role in the transcriptional control of HIF-1α and VEGF. In this dissertation, we demonstrate that both nucleolin and hnRNP K bind selectively to the G- and C-rich sequences, respectively, in the pPu/pPy tract of the HIF-1α and VEGF promoters. Specifically, the small interfering RNA-mediated silencing of either nucleolin or hnRNP K resulted in the down-regulation of basal VEGF gene, and the opposite effect was seen when the transcription factors were overexpressed, suggesting that they act as activators of VEGF transcription. Taken together, the identification of transcription factors that can recognize and bind to atypical DNA structures within pPu/pPy tracts will provide new insight into mechanisms of transcriptional regulation of the HIF-1α and VEGF gene.

The DNA is a flexible and heterogeneous molecule that can adopt different local conformations alternative to the classical double-helix. These noncanonical structures are known as non-B DNAs. These conformers appear to play an important role in different physiological and pathological cellular conditions and influence many biochemical properties of the genome. The formation of these structures is dependent upon specific features of the DNA sequence and different patterns may lead to the formation of different non-B DNAs. Due to lack of updated and flexible computational methods, during these years I focused my work on the development of new tools for the detection of some of these patterns at a genome-wide scale. Particularly, I focused on the detection of patterns that are degenerate. For this task, I developed NeSSie and QPARSE. NeSSie efficiently and exhaustively detects sequences with symmetrical properties, such as mirrors and palindromes that are associated to the formation of hairpins, cruciforms, and triple-stranded DNA. QPARSE detects consecutive exact or degenerate runs of Gs (G-islands) that are involved in the formation of G-quadruplex (G4) and paired G-quadruplex structures, i.e. two quadruplex structures that are close to each other along the sequence and that can fold cooperatively interacting into a higher-order structure. Eventually, I started using these tools to perform analyses on Mycobacterium spp. and human genomes. In the genomes of Mycobacterium spp. that are capable of developing tuberculosis-like diseases, NeSSie revealed the enrichment of a pattern with perfect mirror properties. Experimental analyses confirmed that the pattern can fold into a previously unknown but very stable hairpin structure. In the human genome, I focused on the detection of paired G-quadruplex systems. A genome-wide analysis revealed a striking enrichment of sequences potentially involved in the formation of paired G4 systems in correspondence of the TSS (Transcription Starting Site) of thousands of human genes. Among the predicted systems, one has been detected in correspondence of BCL2 TSS and ongoing experimental validations suggest a cooperative folding of the two G-quadruplex structures. These results contribute to the idea that non-B DNAs can play important functional and potentially structural roles. They also suggest that the folding landscape of the DNA molecule is much more complex than previously assumed, and we have a huge lack of knowledge towards the alternative structures that can form in DNA. Following these evidences, the DNA sequence needs to be widely re-evaluated considering also its structural properties addressing efforts both at computational and experimental validation levels.
La doppia elica del DNA è una molecola molto flessibile ed eterogenea, che può adottare una vasta gamma di conformazioni locali alternative. Queste conformazioni vengono collettivamente chiamate non-B DNA. Questi conformeri sembrano svolgere un ruolo importante in diverse condizioni cellulari sia fisiologiche che patologiche, ed influenzano molte proprietà biochimiche del genoma. La formazione di queste strutture dipende da caratteristiche specifiche della sequenza del DNA, e diversi motivi di sequenza possono portare alla formazione di diverse strutture non-B DNA. Durante questi anni, ho concentrato il mio lavoro sullo sviluppo di nuovi strumenti computazionali per la rilevazione di alcuni di questi motivi su scala genomica. Questo investimento di tempo è stato necessario, poiché attualmente mancano strumenti sufficientemente flessibili in grado di eseguire tali analisi. In particolare, mi sono concentrato sul rilevamento di motivi degenerati. A tale scopo, ho sviluppato NeSSie e QPARSE. NeSSie è in grado di rilevare in modo efficiente ed esauriente sequenze con proprietà simmetriche, come motivi speculari e palindromici associati alla formazione di forcine, strutture cruciformi e regioni di DNA a triplo filamento. QPARSE può rilevare ripetizioni consecutive di isole di G esatte o degenerate, che sono coinvolte nella formazione di G-quadruplex (G4) e strutture G-quadruplex appaiate (cioè due strutture quadruplex che si trovano vicine lungo la sequenza e che possono interagire formando una struttura di ordine superiore ed influenzandosi reciprocamente nel ripiegamento). Ho quindi iniziato a utilizzare questi strumenti per eseguire analisi su genomi appartenenti a specie di micobatterio e sul genoma umano. Nei genomi delle specie di micobatteri che sono in grado di sviluppare malattie simili alla tubercolosi, NeSSie ha rivelato l'arricchimento di un motivo con una perfetta simmetria a specchio. Analisi sperimentali hanno quindi confermato che questo motivo può piegarsi in una struttura a forcina precedentemente sconosciuta ma molto stabile. Nel genoma umano, mi sono concentrato sul rilevamento di sistemi G-quadruplex accoppiati. Una analisi su tutto il genoma ha rivelato un sorprendente arricchimento di sequenze potenzialmente coinvolte nella formazione di questi sistemi in corrispondenza del TSS (Sito di inizio della trascrizione) di migliaia di geni umani. Tra i sistemi predetti, uno identificato in corrispondenza del TSS di BCL2 è in corso di validazione sperimentale e i risultati preliminari sono promettenti. Questi risultati contribuiscono all'idea che i non-B DNA possano svolgere importanti ruoli funzionali e potenzialmente strutturali. Suggeriscono anche che il panorama di strutture che possono formarsi nella molecola di DNA sia molto più complesso di quanto ipotizzato, e che abbiamo ancora un'enorme mancanza di conoscenza verso queste strutture alternative. Seguendo queste evidenze, la sequenza del DNA deve essere ampiamente rivalutata non solo dal punto di vista della codifica, ma considerando anche le sue proprietà strutturali e funzionali. È quindi necessario indirizzare gli sforzi verso nuovi campi di indagine, studiando e caratterizzando queste strutture a livello genomico.