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1
GUARDAMAGNA, ISABELLA. "A new functional in vitro cell-free assay to evaluate DNA repair mechanisms." Doctoral thesis, Università degli studi di Pavia, 2020. http://hdl.handle.net/11571/1301947.
Повний текст джерелаАнотація:
DNA is exposed to endogenous and exogenous agents that are
potential causes of several pathological processes; for this reason,
eukaryotic cells developed many mechanisms able to control and repair
lesions. One of them, Nucleotide Excision Repair (NER) is a highly versatile
and complex system by which UV-photolesions, such as cyclobutane
pyrimidine dimers (CPDs) or pyrimidine (6-4) pyrimidone photoproducts (6-
4PPs), are recognized and removed.
A key factor, involved in the recognition of chromatin photolesions, is
DDB2 (DNA Damaged binding protein 2) thanks to its ability of creating a
complex together with DDB1 (UV-DDB complex). Recently, it was
demonstrated that DDB2 binds directly PCNA (Proliferating Cell Nuclear
Antigen) through a conserved sequence called PIP-box; the disruption of
this binding in the mutated form (DDB2PCNA-) induces a delayed DNA
damage recognition but also an inefficient DNA repair activation.
To better clarify this delay, it was developed a new functional in vitro
cell-free system in which repair activity, in isolated nuclei, was evaluated.
Its responsiveness was also evaluated with different type of DNA lesions,
activating different DNA repair processes, increasing further its
applicability.
Moreover, the involvement of DDB1 was studied as possible actor
when DDB2 loses its function. In the presence of DDB2PCNA- protein, the
DNA repair process is inefficient, nevertheless, not completely blocked. For
this reason, it was hypothesized a possible ability of DDB1 to bind directly
PCNA, when DDB2 is altered or ineffective.
2
Liton, Kumar Saha. "Differential Micronucleus Frequency in Isogenic Human Cells Deficient in DNA Repair Pathways Is a Valuable Indicator for Evaluating Genotoxic Agents and Their Genotoxic Mechanisms." Kyoto University, 2019. http://hdl.handle.net/2433/242428.
Повний текст джерелаАнотація:
付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラムKyoto University (京都大学)
0048
新制・課程博士
博士(医科学)
甲第21696号
医科博第100号
新制||医科||7(附属図書館)
京都大学大学院医学研究科医科学専攻
(主査)教授 齊藤 博英, 教授 清水 章, 教授 Shohab YOUSSEFIAN
学位規則第4条第1項該当
3
Ruiz, Alarcón Rafael. "Targeting DNA repair mechanisms in aggresive neuroblastoma." Thesis, Högskolan i Skövde, Institutionen för hälsovetenskaper, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-19821.
Повний текст джерелаАнотація:
Neuroblastoma is a tumour derived from cells of the nervous system and is the most common solid tumour in childhood. MYCN amplified and 11q-deleted neuroblastoma, two high-risk neuroblastoma were investigated in this study. RAD51 gene family includes six central genes for the dsDNA breaks repair by homologous recombination, which has been reported as important in varying types of cancer. The study aims to investigate if the dysregulation of this gene family could be involved in the unstable genome of 11q-deleted neuroblastoma, and to better understand the link between both high-risk tumours. The RAD51 family genes’ expression level was measured by RT-qPCR in samples of 11q-deleted and MYCN-amplified neuroblastoma that were treated with a UVC treatment and were recovered during varying hours. R2 database and DAVID were used to study the RAD51 family’s expression levels, associated event-free survivability, and altered pathways. RAD51 family is highly dysregulated in these tumours, four genes of six were found to be altered in high-risk neuroblastoma. Four of six genes presented altered expression levels in 11q-loss, and three of six in the MYCN-amplified case after the UVC treatment. The event-free survival probability analysis shown that the levels of expressions associated with high-risk neuroblastoma coincide with those that represent a poor life expectancy. Altered pathways were different in each type of tumour. 11q-deletion neuroblastoma’s pathways were associated with the nervous system development, and MYCN-amplified was related to the immune system. This study suggests that 11q-loss neuroblastoma presents a greater RAD51 family dysregulation compared with MYCN-amplified one, which could explain why its genome is unstable.
4
Gessner, Sophia Johanna. "Molecular mechanisms of DNA repair in Mycobacterium tuberculosis." Doctoral thesis, University of Cape Town, 2017. http://hdl.handle.net/11427/26861.
Повний текст джерелаАнотація:
The mycobacterial DNA damage and repair pathways involved in the emergence of drug-resistance during host infection remain poorly understood, yet are critical to any efforts to develop novel "anti-evolution" drugs aimed at reducing the capacity of Mycobacterium tuberculosis to adapt genetically during tuberculosis (TB) treatment. The thesis presented here aimed to investigate the contribution of the DNA damage (SOS) response in adaptive mutagenesis, and focused on two specific components: the role of the specialist translesion synthesis DNA polymerase, DnaE2, in mutagenesis under stress and, secondly, the function of the mycobacterial homologue of a putative SOS response associated peptidase (SRAP) protein which has been identified in comparative genomics analyses of organisms possessing a DnaE2-type C family DNA polymerase. This work focused on the putative SRAP protein which was predicted to form part of the mycobacterial DNA damage response as a functional switch by binding DNA in an autoproteolytic dependent manner. To this end, SRAP deletion mutants were generated for both M. smegmatis (MSMEG_1891) and M. tuberculosis (Rv3226c). Despite the fact that SRAP was upregulated in both M. smegmatis and M. tuberculosis following genotoxic stress, no DNA damage phenotype was detected in any SRAP deletion mutant using a variety of DNA damaging agents. In parallel, an eGFP-tagged M. smegmatis SRAP allele was constructed to enable visualisation of SRAP upregulation and sub-cellular recruitment using fluorescent microscopy; however no eGFP expression could be visualised after MMC treatment. It was not clear whether this was due to faulty eGFP expression in the fusion protein, or to low-level induction of SRAP. In a biochemical approach to elucidate SRAP function, soluble M. smegmatis SRAP protein was expressed and purified using a N-terminal hexa-histidine tag. No proteolytic activity was detected in gelatine or casein zymography, perhaps indicating that SRAP has a very specific substrate. Moreover, while it was predicted that autocatalytic cleavage of the C-terminus was required for activation of SRAP, no such cleavage was detected using hexa-histidine tag staining, possibly pointing to a set of very specific conditions for activation. In combination, therefore, neither microbiological nor biochemical assays could elucidate a definitive role for SRAP in the mycobacterial DNA damage response. DnaE2 has been directly implicated in induced mutagenesis to rifampicin (Rif) resistance in Mycobacterium tuberculosis following exposure of bacilli to genotoxic stress. In previous work in our group, a vitamin B₁₂-sensitive ΔmetH strain was found to form "B₁₂-resistant" suppressor mutants at a frequency higher than could be explained by spontaneous mutagenesis alone. The first part of this thesis investigated the potential role of DnaE2 in the high-frequency emergence of B₁₂-resistance by mutating DnaE2 in the ΔmetH background. Whereas elimination of polymerase function in a DnaE2ᴬᴵᴬ mutant abrogated DNA damage-induced mutagenesis to Rif resistance, no change in B₁₂ sensitivity was detected in a ΔmetH dnaE2ᴬᴵᴬ double mutant. PCR sequencing of spontaneous B₁₂-resistant mutants revealed mutations in genes previously associated with the suppressor phenotype; moreover, there was no apparent difference in the nature of mutations observed in both parental and dnaE2ᴬᴵᴬ mutant strains. Instead, these results suggest that an alternative mechanism must exist to enable adaptive mutagenesis in methionine-starved mycobacteria.
5
Malik, Shivani. "REGULATORY MECHANISMS OF TRANSCRIPTION AND ASSOCIATED DNA REPAIR." OpenSIUC, 2012. https://opensiuc.lib.siu.edu/dissertations/626.
Повний текст джерелаАнотація:
Transcription is a crucial regulatory step in gene regulation modulated by several proteins. Any misregulation during transcription can lead to many diseases including cancer, neurodegenerative disorders and aging making it imperative to have a detailed mechanistic view of the process. Over the recent years, 26S proteasome has been implicated in transcriptional regulation through its proteolytic and non-proteolytic activities. While, the proteolytic role of proteasome in transcription has been extensively studied, its non-proteolytic function is poorly understood. Thus, one of my thesis aims had been to analyze the non-proteolytic role of proteasome in transcription. My results have revealed the non-proteolytic role of 26S proteasome in establishing a specific protein interaction network at the promoter for stimulated transcriptional initiation in vivo . In addition to its roles in transcription, 26S proteasome also plays an important role in the degradation of RNA polymerase II stalled at DNA lesion facilitating the rapid repair of transcriptionally active genes through a process of transcription coupled repair (TCR). My studies have addressed the key question of the fate of RNA polymerase II stalled at a lesion. My findings show that RNA polymerase II interacts with an elongation and TCR-specific factor, Rad26p. Upon encountering a lesion, RNA polymerase II stalls and unloads Rad26p on the site of DNA damage. Subsequently, the elongating RNA polymerase II is disassembled through the degradation of its largest subunit, Rpb1p. Further; our studies have also uncovered a novel role of Rad26p in chromatin disassembly, which facilitates transcriptional elongation and hence TCR. This work provides valuable insights into interplay of chromatin structure, transcriptional elongation and TCR. Finally, extending the regulatory knowledge of sense transcriptional initiation to antisense, my work has revealed the extensive participation of GTFs in the process. Collectively, results of above studies provide a comprehensive view of transcription and associated process of active genome repair.
6
Shell, Scarlet Sara. "Mechanisms of initiation of DNA mismatch repair in Saccharomyces cerevisiae." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p3307558.
Повний текст джерелаАнотація:
Thesis (Ph. D.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed July 23, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
7
De, Silva Inusha Udanie. "Mechanisms of repair of DNA damage produced by antitumour drugs." Thesis, University College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.404490.
Повний текст джерела
8
Cannan, Wendy J. "Mechanisms and Dynamics of Oxidative DNA Damage Repair in Nucleosomes." ScholarWorks @ UVM, 2016. http://scholarworks.uvm.edu/graddis/628.
Повний текст джерелаАнотація:
DNA provides the blueprint for cell function and growth, as well as ensuring continuity from one cell generation to the next. In order to compact, protect, and regulate this vital information, DNA is packaged by histone proteins into nucleosomes, which are the fundamental subunits of chromatin. Reactive oxygen species, generated by both endogenous and exogenous agents, can react with DNA, altering base chemistry and generating DNA strand breaks. Left unrepaired, these oxidation products can result in mutations and/or cell death. The Base Excision Repair (BER) pathway exists to deal with damaged bases and single-stranded DNA breaks. However, the packaging of DNA into chromatin provides roadblocks to repair. Damaged DNA bases may be buried within nucleosomes, where they are inaccessible to repair enzymes and other DNA binding proteins. Previous in vitro studies by our lab have demonstrated that BER enzymes can function within this challenging environment, albeit in a reduced capacity.
Exposure to ionizing radiation often results in multiple, clustered oxidative lesions. Near-simultaneous BER of two lesions located on opposing strands within a single helical turn of DNA of one another creates multiple DNA single-strand break intermediates. This, in turn, may create a potentially lethal double-strand break (DSB) that can no longer be repaired by BER. To determine if chromatin offers protection from this phenomenon, we incubated DNA glycosylases with nucleosomes containing clustered damages in an attempt to generate DSBs. We discovered that nucleosomes offer substantial protection from inadvertent DSB formation. Steric hindrance by the histone core in the nucleosome was a major factor in restricting DSB formation. As well, lesions positioned very close to one another were refractory to processing, with one lesion blocking or disrupting access to the second site. The nucleosome itself appears to remain intact during DSB formation, and in some cases, no DNA is released from the histones. Taken together, these results suggest that in vivo, DSBs generated by BER occur primarily in regions of the genome associated with elevated rates of nucleosome turnover or remodeling, and in the short linker DNA segments that lie between adjacent nucleosomes.
DNA ligase IIIα (LigIIIα) catalyzes the final step in BER. In order to facilitate repair, DNA ligase must completely encircle the DNA helix. Thus, DNA ligase must at least transiently disrupt histone-DNA contacts. To determine how LigIIIα functions in nucleosomes, given this restraint, we incubated the enzyme with nick-containing nucleosomes. We found that a nick located further within the nucleosome was ligated at a lower rate than one located closer to the edge. This indicated that LigIIIα must wait for DNA to spontaneously, transiently unwrap from the histone octamer to expose the nick for recognition. Remarkably, the disruption that must occur for ligation is both limited and transient: the nucleosome remains resistant to enzymatic digest before and during ligation, and reforms completely once LigIIIα dissociates.
9
Shivji, Mahmud K. K. "Nucleotide excision repair of DNA : dissection and reconstitution in vitro." Thesis, Open University, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309860.
Повний текст джерела
10
Shen, Ying. "Studies on the mechanisms of RNA-driven DNA repair and modification." Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/45969.
Повний текст джерелаАнотація:
Our previous studies have demonstrated that RNA can serve as a template for double-strand break (DSB) repair in the yeast Saccharomyces cerevisiae using synthetic RNA-containing oligonucleotides (oligos). Following this initial work, we show that the RNA tract of RNA-containing oligos can be copied into DNA to transfer a genetic change at the chromosomal level also in the bacterium Escherichia coli and in human cells. Exploiting the use of oligos containing ribonucleoside monophosphates (rNMPs), we developed a molecular approach to generate RNA/DNA hybrids of chosen sequence and structure at the chromosomal level in both yeast and E. coli cells. Such technique allows us to study how rNMPs present in the DNA genome of cells are tolerated by cells, what factors recognize and target rNMPs in DNA and to what extent the embedded rNMPs may alter genome integrity. Here we proved that mispaired rNMPs embedded into genomic DNA, if not removed, serve as templates for DNA synthesis during chromosomal replication and produce a genetic change. We discovered that mispaired rNMPs that are embedded in genomic DNA are not only targeted by ribonucleases H (RNases H) but also by the mismatch repair (MMR) system both in yeast and in E. coli. Our data reveal novel substrates for the MMR system, and also uncover an unpredicted competition between RNase H and MMR for the RNA/DNA mispairs.
11
Fiala, Kevin Andrew. "A kinetic and biochemical approach to understanding the mechanisms of novel DNA polymerases." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1187048005.
Повний текст джерела
12
Nichols, Joseph A. "In vitro binding of base excision repair glycosylases to poly(adp-ribose)." Online access for everyone, 2008. http://www.dissertations.wsu.edu/Thesis/Summer2008/j_nichols_071008.pdf.
Повний текст джерела
13
Zhang, Hongshan. "A single molecule perspective on DNA double-strand break repair mechanisms." Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0177.
Повний текст джерелаАнотація:
Les cassures double brin de l'ADN altèrent l'intégrité physique du chromosome et constituent l'un des types les plus sévères de dommages à l'ADN. Pour préserver l'intégrité du génome contre les effets potentiellement néfastes des cassures double brin de l'ADN, les cellules humaines ont développé plusieurs mécanismes de réparation, dont la réparation par recombinaison de l'ADN et la jonction d'extrémités non-homologues (NHEJ), catalysés par des enzymes spécifiques. Pendant ma thèse, nous avons caractérisé la dynamique de certaines des interactions protéines/ADN impliquées dans ces mécanismes au niveau de la molécule unique. Dans ce but, nous avons combiné des pinces optiques et de la micro-fluidique avec de la microscopie de fluorescence à champ large afin de manipuler une ou deux molécules d'ADN individuelles et d'observer directement les protéines de la réparation marquées par fluorescence agissant sur l'ADN. Nous avons concentré notre analyse sur trois protéines/complexes essentiels impliqués dans la réparation de l'ADN: (i) la protéine humaine d’appariement de brin RAD52, (ii) les protéines humaines XRCC4, XLF et le complexe XRCC4/Ligase IV de la NHEJ et (iii) le complexe humain MRE11/RAD50/NBS1DNA double-strand breaks disrupt the physical continuity of the chromosome and are one of the most severe types of DNA damage. To preserve genome integrity against the potentially deleterious effects of DNA double-strand breaks, human cells have evolved several repair mechanisms including DNA recombinational repair and Non-Homologous End Joining (NHEJ), each catalyzed by specific enzymes. In this thesis we aimed at unraveling the dynamics of protein/DNA transactions involved in DNA double-strand break repair mechanisms at single molecule level. To do this, we combined optical tweezers and microfluidics with wide-field fluorescence microscopy, which allowed us to manipulate individual DNA molecules while directly visualize fluorescently-labeled DNA repair proteins acting on them. We focused the study on three crucial proteins/complexes involved in DNA repair: (i) the human DNA annealing protein RAD52, (ii) the non-homologous end joining human proteins XRCC4 and XLF and the complex XRCC4/Ligase IV, and (iii) the human MRE11/RAD50/NBS1 complex
14
Hall, Ashley. "ASSESSMENT AND IN VITRO REPAIR OF DAMAGED DNA TEMPLATES FROM FORENSIC STAINS." Doctoral diss., University of Central Florida, 2005. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2621.
Повний текст джерелаАнотація:
DNA extracted from biological stains is often intractable to analysis. This may due to a number of factors including a low copy number (LCN) of starting molecules, the presence of soluble inhibitors or damaged DNA templates. Remedies may be available to the forensic scientist to deal with LCN templates and soluble inhibitors but none presently exist for damaged DNA. In fact, only recently has the biochemical nature, the extent of DNA damage in physiological stains and the point at which the damage inflicted upon a particular sample precludes the ability to obtain a genetic profile for purposes of identification been examined. The primary aims of this work were first to ascertain the types of DNA damage encountered in forensically relevant stains, correlating the occurrence this damage with the partial or total loss of a genotype, and then to attempt the repair of the damage by means of in vitro DNA repair systems. The initial focus of the work was the detection of damage caused by exogenous, environmental sources, primarily UV irradiation, but also factors such as heat, humidity and microorganism growth. Results showed that the primary causes of the damage that resulted in profile loss were strand breaks, both single and double stranded, as well as modifications to the DNA structure that inhibited its amplification. Armed with this knowledge, the next focus was the repair of the damage by means of in vitro DNA systems. Efforts have been concentrated on single strand break/gap repair and translesion synthesis assays. By modifying the assays and employing various combinations of the systems, a genetic signature has been recovered from previously intractable samples. Additionally, the effects that various storage conditions have on the DNA in physiological stains stored in a laboratory were examined. The optimal long term storage conditions for biological evidence has been a matter of debate in the forensic community for some time. But, no comprehensive study had previously been undertaken to describe the effects of dehydration and temperature on degradation and the ability to obtain a genetic profile on bloodstains kept in different types of storage media at a range of temperatures. To examine this, bloodstains were either allowed to dry overnight or placed in the storage medium while still wet and were stored at room temperature, 4oC or 30oC for up to four years. Results showed that specimens dehydrated prior to storage were very stable, and these bloodstains showed no degradation or loss of a genetic profile for up to four years.Ph.D.
Department of Chemistry
Arts and Sciences
Biomolecular Sciences: Ph.D.
15
Naom, Isam Said. "Molecular and functional analysis of the sbcC gene of Escherichia coli K12." Thesis, University of Nottingham, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294029.
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Sharples, Gary John. "Molecular organisation and functional analysis of the chromosomal ruv region of Escherichia coli K12." Thesis, University of Nottingham, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.276350.
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McKelvey, V. J. "The role of thymidine kinase in DNA repair processess in cultured mammalian cells." Thesis, University of Ulster, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.378737.
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Chen, Shuhua. "Multiple mechanisms regulate the human replication factors : replication protein A and DNA polymerase alpha-during DNA replication and DNA repair /." [S.l. : s.n.], 2003.
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19
Martin, Paul Ryan. "Studies on the Primary Mechanisms of (6-4) photolyase : Photoactivation and DNA Repair." Thesis, Paris 6, 2014. http://www.theses.fr/2014PA066375/document.
Повний текст джерелаАнотація:
Ce travail concerne les mécanismes photoinduits d’une flavoprotéine appartenant à la famille des cryptochromes et photolyases (CPF) : la photolyase (6-4). En utilisant de la lumière bleue cette protéine répare un dommage de l’ADN induit par les UV, le photoproduit (6-4). Nous avons étudié cette photoreparation ainsi qu’une autre réaction photoactivée que la photolyase utilise pour réduire son cofacteur flavine, la photoactivation. Nous avons faits nos études en utilisant la photolyase (6-4) de Xenopus laevis. Nous avons étudié la photoréduction du chromophore FADox de l’enzyme par spectroscopie d’absorption transitoire femtoseconde polarisée. Nous avons observé un transfert d’électron ultrarapide (~400 fs) après excitation du chromophore FADox. Nous avons caractérisés un résidu tryptophane comme réducteur. Nous avons cherché de distinguer entre les différents tryptophanes présents dans le site de photoactivation par des mesures d’anisotropie. Les résultats obtenus suggèrent que le mécanisme de transfert d’électron dans la photolyase n’est pas compatible avec le mécanisme supposé chez les photolyases qui consiste des transferts d’électron de long d’une chaine de trois résidus tryptophane. Grâce à des trains d’impulsions courtes, nous avons démontré que la photolyase (6-4) répare l’ADN par un mécanisme à deux photons successif. Le premier photon sert à convertir le défaut (6-4) en un intermédiaire métastable, X, qui a une durée de vie de ~2 min. Un second photon absorbé pendant cette durée de vie permet d’achever le cycle de réparationWe studied the light-induced reactions of the (6-4) photolyase, a flavoenzyme of the cryptochrome/photolyase family that repairs the UV-induced (6-4) photodamage in DNA with the aid of blue light. We studied this photorepair reaction as well as the light-induced cofactor reduction called photoactivation that the enzyme uses to bring itself to a repair-active state in the (6-4) photolyase from Xenopus laevis. We have studied the photoactivation of the FADox cofactor of the enzyme using femtosecond polarised transient absorption spectroscopy. We observed a sub-picosecond electron transfer (~400 fs) after excitation of the FADox cofactor. We were able to characterise a tryptophan residue as the electron donor. We sought to differentiate the spectroscopically identical but differently oriented tryptophan residues within the protein’s photoactivation site by transient anisotropy measurements. Our results suggest that the photoactivation mechanism is not fully compatible with the mechanism thought to be conserved among photolyases: an electron transfer mechanism via electron hopping along a chain of three highly conserved tryptophan residues.Using series of single turnover flashes, we have found that the repair reaction proceeds by a successive two-photon mechanism. The first photon converts the (6-4) lesion into a metastable intermediate X, the lifetime of which is ~2 min. Absorption of a second photon within the lifetime of X results to the restoration of intact nucleobases. In light of our findings, the reaction was also studied by femtosecond transient absorption spectroscopy
20
Rosales, HernaÌndez Alma L. "Mechanisms of β cell DNA damage and repair in type 1 diabetes mellitus." Thesis, University of Brighton, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.247817.
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Jain, Rupal. "Understanding DNA Repair and Damage-Tolerance Mechanisms in the Hyperthermophilic Crenarchaeote Sulfolobus acidocaldarius." University of Cincinnati / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1575967480086557.
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22
Barrett, Aaron James. "Roles and mechanisms of DNA repair factors and pathways in maintaining seed quality." Thesis, University of Leeds, 2015. http://etheses.whiterose.ac.uk/11489/.
Повний текст джерелаАнотація:
Successful germination is a major determinant of crop yields and survival of plants in the natural environment. Our knowledge of molecular factors important to seed quality is far from complete, yet such understanding is vital in the endeavour of mitigating the detrimental effects of extended storage on seed vigour and viability. Genome integrity is crucial for cellular survival and transmission of genetic information. A number of pre-genomic era studies identified strong correlations between DNA damage accumulated in the quiescent seed and seed ageing. If unrepaired, DNA damage results in delayed growth, mutagenesis and cell death. Damage products incurred by DNA are remarkably heterologous and plants have evolved multiple pathways to facilitate the repair of specific damage products. Through the isolation and analysis of knockout Arabidopsis mutants, this work identifies and characterises DNA repair genes whose action is required during seed imbibition. These studies examined the importance of four major DNA repair pathways in germination and seed quality. Mutants deficient in non-homologues end joining (KU70 and KU80), homologues recombination (XRCC2) and base excision repair (ARP) DNA repair pathways were all found to be hypersensitive to accelerated ageing treatment but those deficient in nucleotide excision repair (ERCC1) were not. Therefore this work establishes roles for multiple DNA repair pathways in seed longevity. Comparative analysis also defined non-homologues end joining as the most important DNA repair pathway to seed quality. LIG6 encodes a unique plant specific DNA ligase with roles in seed longevity and implicated in repair of DSBs but remains largely uncharacterised to date. Here studies using extra-chromosomal recombination assays demonstrate LIG6 functions in the promotion of recombination activities in Arabidopsis protoplasts. Further studies demonstrate the involvement of LIG6 in the maintenance of root meristem genome stability. Collectively, this work provides an increased understanding of the early events central to the germination process and seed longevity.
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Gopaul, Diyavarshini. "Study of the molecular mechanisms linking transcription and DNA repair in Saccharomyces cerevisiae." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS347.
Повний текст джерелаАнотація:
La voie de réparation par excision de nucléotides (NER) répare les lésions qui distordent la double hélice d’ADN notamment ceux induits par l’irradiation UV. Le NER est subdivisé en deux sous-voies : GG-NER (Global Genome Repair) et TC-NER (Transcription-Coupled Repair). La sous-voie GG-NER enlève les dommages à l’ADN dans l’ensemble du génome. La sous-voie TC-NER répare les dommages sur le brin transcrit qui interfèrent avec la progression de l’ARN Pol II. Les défauts de la voie NER peuvent conduire à l’apparition de pathologies graves. Par exemple, des mutations dans le gène XPG, codant une 3’ endonucléase impliquée dans la voie NER, peuvent mener au xeroderma pigmentosum (XP) associé ou non au syndrome de Cockayne (CS).Récemment, le laboratoire a découvert un lien fonctionnel entre Rad2, homologue chez la levure Saccharomyces cerevisiae de la protéine XPG humaine, et le Médiateur (Eyboulet et al., 2013). Le Médiateur est un complexe multiprotéique nécessaire à la régulation de la transcription dépendante de l’ARN Pol II. Cette étude a suggéré que le Médiateur est impliqué dans la sous-voie TC-NER en facilitant le recrutement de Rad2 au niveau des régions transcrites.Mon projet de thèse visait à étudier les mécanismes moléculaires qui lient la transcription et la réparation de l’ADN. Plus précisément, d’investiguer le lien fonctionnel entre le Médiateur et la machinerie du NER chez S. cerevisiae.Lors du TC-NER, l’ARN Pol II est le premier facteur signalant le dommage à l’ADN. De plus, le Médiateur et Rad2 interagissent avec l’ARN Pol II. Pour déterminer le lien fonctionnel entre ces composants, nous avons utilisé des approches de génétique et génomique dans les mutants de TFIIH (kin28), de l’ARN Pol II (rpb9) and du Médiateur (med17). Nos résultats nous ont permis de proposer un modèle dans lequel Rad2 est recruté au niveau des régions régulatrices enrichies par le Médiateur, et Rad2 est ensuite transféré au niveau des régions transcrites de manière dépendante à l’ARN Pol II. De plus, ces résultats suggèrent que le rôle du Médiateur dans la transcription est fortement lié à son rôle dans la réparation de l’ADN.Ensuite, nous avons montré que le lien entre le Médiateur et la machinerie du NER peut être étendu à d’autres protéines du NER notamment en démontrant une interaction physique entre le Médiateur et Rad1/XPF, Rad10/ERCC1 ou Rad26/CSB, en l’absence des UV. Tout comme Rad2, nous avons démontré que Rad1 et Rad10 n’ont pas de rôle majeur dans la transcription. Pour approfondir le lien entre ces protéines du NER et le Médiateur, des expériences de ChIP-sequencing ont été réalisées. Nous avons observé que le Médiateur est présent au niveau de certaines régions qui sont aussi enrichies par ces protéines du NER. Après l’induction des dommages par UV, les interactions entre le Médiateur et la machinerie du NER reste inchangées par rapport aux conditions en l’absence des UV. De plus grâce à nos expériences de ChIP, nous avons observé un changement de la liaison à la chromatine des protéines du NER et du Médiateur après l’irradiation aux UV. Des expériences de ChIP-sequencing seront réalisées pour avoir une vue globale de ces changements.En conclusion, nous avons solidifié le lien fonctionnel entre Rad2, le Médiateur et l’ARN Pol II par rapport à la réparation couplée à la transcription. Nous avons aussi démontré que le Médiateur interagit avec d’autres protéines du NER (Rad1/XPF, Rad10/ERCC1 et Rad26/CSB) et colocalise avec eux sur certaines régions de la chromatine. En somme, notre projet place le Médiateur à l’interface de la transcription et de la réparation de l’ADN, deux processus essentiels dont les défauts peuvent mener à des pathologies gravesNucleotide excision repair (NER) is a well conserved pathway that removes helix-distorting DNA lesions such as those arising upon UV irradiation. Global genome repair subpathway (GG-NER) removes the DNA lesions in the genome overall, and transcription-coupled repair (TC-NER) removes the DNA lesions interfering with Pol II progression through actively-transcribed regions. Defects in the NER pathway may lead to severe human pathologies. For instance, mutations in human XPG gene, encoding a 3’ endonuclease essential for NER, give rise to xeroderma pigmentosum (XP) sometimes associated with Cockayne syndrome (CS). Recently, the laboratory discovered a functional link between Rad2/XPG and Mediator in Saccharomyces cerevisiae (Eyboulet et al., 2013). Mediator is a large multisubunit complex essential for transcription regulation. We suggest that Mediator is involved in TC-NER by facilitating Rad2 recruitment to transcribed genes.My PhD work aimed at addressing the molecular mechanisms of this link between transcription and DNA repair, especially by investigating the functional interplay between Mediator and the NER machinery in yeast Saccharomyces cerevisiae.RNA Pol II is the first complex of TC-NER that encounters the DNA damage. Moreover, both Mediator and Rad2/XPG interact with Pol II. However, a functional interplay between all these components related to TC-NER remained to be determined. Using genetic and genomic approaches, in particular ChIP-sequencing in TFIIH (kin28), RNA Pol II (rpb9) and Mediator (med17) mutants, our work led us to propose a model where Rad2 shuttles between Mediator on upstream activating sequence (UAS) and RNA Pol II on transcribed regions (Georges, Gopaul et al., under review). Our results also suggest that Mediator functions in transcription and DNA repair are closely related.Moreover, we showed that Mediator’s link to NER can be extended to other NER proteins. Indeed, we identified a physical interaction between Mediator and other NER proteins, including Rad1/XPF, Rad10/ERCC1 and Rad26/CSB in the absence of UV irradiation. Similarly to Rad2, we demonstrated that Rad1 and Rad10 do not have a major role in yeast transcription. To further study the functional link between Mediator and the NER machinery, we obtained the genomic distribution of different NER proteins by ChIP-sequencing. We found that some promoter regions are co-occupied by Mediator and these NER proteins, and that relationships between Mediator and these NER proteins are more complex than between Mediator and Rad2. We also investigated if physical interactions between Mediator and NER proteins are modified after UV, we did not observe any significant change. Furthermore, we observed that the chromatin binding profiles of NER proteins and Mediator are modified after UV-irradiation. ChIP-sequencing will be carried out to get a genome-wide view of their chromatin binding profiles.In conclusion, we have strengthened the link between Rad2/XPG, Mediator and RNA Pol II, providing mechanistic insights into functional interplay between these components related to transcription-coupled repair, and showed that the link between Mediator and the NER machinery can be extended to other proteins. Taken together, our results suggest a close relation between Mediator functions in transcription and in NER, two fundamental processes dysfunction of which leads to human diseases
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Adams, Bret. "Double-Strand Break Repair Mechanisms in Human Embryonic Stem Cells." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/114.
Повний текст джерелаАнотація:
Central to the progression of all organisms is the maintenance of a stable genome despite continuous insults arising from genotoxic and environmental stresses. Embryonic stem cells show promise for treatment of a variety of diseases as well as for providing normal human tissue to conduct scientific research. A major obstacle for their application is that genomic instability arises in stem cells after prolonged cell culture. The most detrimental form of DNA damage is the DNA double-strand break (DSB), which is managed by cells through complex mechanisms, designated the DNA damage response. There are two major types of DSB repair; homologous recombination repair (HRR) and non-homologous end joining (NHEJ), both of which are regulated by members of the phosphatidyl-inositol-3’-kinase-related kinase (PIKK) family, including Ataxia Telangiectasia Mutated (ATM), Ataxia Telangiectasia Mutated and Rad3-related (ATR) and the DNA dependent protein kinase (DNA-PK). The aim of this study was to define the mechanisms and important proteins involved in repair of human embryonic stem cells. Here we have also described a system to differentiate hESCs into neural progenitors and astrocytes and were able to examine their DNA damage response. In both examining DNA repair markers and using a DNA repair reporter assay, this work shows that ATR is involved in DSB repair early in development, whereas ATM is essential in DSB repair in differentiated cells. We also show that HRR, a high fidelity form of repair, is used extensively by embryonic stem cells and HRR diminishes as cells differentiate. We also further defined the extent of NHEJ and the role of high fidelity NHEJ from the embryonic to differentiated state. These findings further the basic knowledge of repair fidelity in embryonic and mature human tissue. The data gives insight into what proteins maintain stem cell genomic stability and may be important to develop safe technologies for tissue engineering. Specifically, we have defined what DNA damage signaling pathways are used as embryologic cells progress to a mature, functional state.
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Foote, Maha Zewail. "The antitumor agent ecteinascidin 743 (Et 743) characterization of its covalent DNA adducts and its effect on DNA repair mechanisms /." Digital version:, 2000. http://wwwlib.umi.com/cr/utexas/fullcit?p9992788.
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Showalter, Alexander Keith. "KINETIC STUDIES OF TWO ERROR-PRONE DNA REPAIR ENZYMES: POSSIBLE MECHANISMS FOR VIRAL MUTAGENESIS." Connect to this title online, 2002. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1016207119.
Повний текст джерелаАнотація:
Thesis (Ph. D.)--Ohio State University, 2002.Title from first page of PDF file. Document formatted into pages; contains xii, 97 p.; also contains graphics (some col.). Includes abstract and vita. Advisor: Ming-Daw Tsai, Dept. of Chemistry. Includes bibliographical references (p. 92-97).
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Stephanou, Nicolas Constantinos. "Mycobacterial non-homologous end-joining : molecular mechanisms and components of a novel DNA double strand break repair pathway /." Access full-text from WCMC, 2008. http://proquest.umi.com/pqdweb?did=1528973431&sid=21&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Zhang, Meng. "Redox Tuning of Flavin and Ultrafast Electron Transfer Mechanisms in DNA Repair by Photolyases." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469101235.
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Zhang, Qingbei. "MOLECULAR MECHANISMS THAT MEDIATE METASTASIS SUPPRESSOR ACTIVITY OF NM23-H1." UKnowledge, 2006. http://uknowledge.uky.edu/gradschool_diss/410.
Повний текст джерелаАнотація:
Metastasis is the spread of cancer cells from the primary tumor to distant sites. It is the most dangerous attribute of cancer, and also the principle cause of cancerrelated morbidity and mortality. Metastasis suppressor genes are a group of genes that suppress tumor metastasis without significant effect on tumorigenicity. NM23 was the first identified metastasis suppressor gene, and loss of its expression is a frequent hallmark of metastatic growth in multiple cancers (e.g. melanoma, carcinomas of breast, stomach and liver). NM23-H1 possesses at least three enzymatic activities, including nucleoside diphosphate kinase (NDPK), histidine kinase (hisK), and a more recently described 3f-5f exonuclease (EXO). While the hisK has been shown to be linked to the suppression of cell motility, the NDPK has been reported to be unrelated to the suppression of metastatic potential indirectly. Relevance of EXO has not been addressed. Other known 3f-5f exonuclease are closely associated with DNA repair functions, suggesting NM23-H1 may suppress mutations required for metastasis. As a transcription factor, NM23 has been shown to modestly downregulate the transcription on PDGF-A chain, a growth factor oncogene, either alone or in association with another transcriptional factor, Pur@. At the same time, identification of NM23-H1 as a 3f-5fexonuclease suggests the role of NM23-H1 in DNA repair. Etoposide and cisplatin elicited nuclear translocation of H1 within 4 h in HeLa and HepG2 cells, seen as accumulation of H1 in small intranuclear foci, strongly suggesting the DNA repair function of H1. To investigate the enzymatic function contributing to metastasis suppressor activity of H1, complementation system was used by transfecting NM23-H1 with individually disrupted enzymatic function into 2 melanoma cell lines, 1205LU and WM793. Overexpression of H1 in 1205LU suppressed lung metastasis in vivo without effect on indices of transformation (e.g. proliferation, soft agar colonization). EXO- deficient H1 and NDPK-deficient H1 lost suppression of lung metastasis, while hisK-deficient H1 maintained suppressor activity. Consistent with the results in 1205LU cells, EXO-deficient H1 and NDPKdeficient H1 lost suppression of the progression of WM793 cells in protein-free medium, while WT and hisK-deficient H1 prevented the progression. Taken together, these data suggest that the NDPK and/or 3f-5fEXO activity of H1 inhibits the progression of premetastatic cells to the metastatic phenotype, possibly via a DNA repair function or other structural transactions with DNA.
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Sakofsky, Cynthia J. "Mechanisms Of Genome Stability In The Hyperthermophilic Archaeon Sulfolobus acidocaldarius." University of Cincinnati / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1321370182.
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Thientosapol, Sanchai. "Mechanisms of transversion mutation are dependent on sequence context and nucleotide paucity during antibody somatic hypermutation." Thesis, The University of Sydney, 2018. http://hdl.handle.net/2123/20061.
Повний текст джерелаАнотація:
Somatic hypermutation of antibodies during humoral immune responses depends on expression of Activation Induced Deaminase (AID) in antibody-producing B cells. AID initiates somatic hypermutation by converting cytosine (C) residues in antibody genes into uracil (U) residues, by deamination. Alone, conversion of cytosine into uracil can only produce C:G to T:A transition mutations, by replication across U (phase 1A mutation). Processing of C deaminations by base excision repair (BER) or mismatch repair (MMR) diversifies mutation, predominantly at C:G (phase 1B mutation) and A:T (phase 2 mutation), respectively. Mutations at C along the Ig variable region are not equally distributed. AID de-aminates C more often if they occur as part of WRCY motif (A/T,A/G,C,C/T). WRCY sequences are concentrated in hypervariable regions of Ig genes, where nucleotide substitutions are likely to be effective at generating useful amino acid substitutions to optimize affinity maturation. Of all WRCY motifs, AGCT and AACT are the most mutated hotspots. AGCT is also enriched in switch regions and facilitates CSR. In Chapter three, using large datasets of a transgenic mouse model, I compared Igh hypermutation between SWHEL B cells, SWHEL B cells deficient for UNG2 via retroviral expression of the uracil glycosylase inhibitor (ugi), SWHEL B cells deficient for MutSα by crossing Msh2ko alleles into SWHEL mice and SWHEL B cells deficient for both UNG2 and MutSα. I found that phase 1B mutations occur by distinct MMR-independent or MMR dependent pathways. At or in proximity to AGCW motifs, phase 1B mutations were driven by UNG2 without requirement for mismatch repair. Deaminations in AGCW were refractive both to processing by UNG2 and to high-fidelity base excision repair (BER) downstream of UNG2, regardless of mismatch repair activity. Outside AGCW motifs, transversions at C:G are co-dependent on UNG2 and MMR. Classically, MMR mediates high fidelity repair of mismatches introduced during replication. The reasons for the profound differences in repair accuracy between classical and AID-induced MMR have not been elicited. During S-phase of the cell replication cycle, when classical post-replication MMR occurs, nucleotide triphosphate (dNTP) levels are optimal for DNA replication, while in G1-phase dNTP levels are lower. Since there is evidence that AID is active in G1-phase, we hypothesized that low dNTP levels may be the cause of low fidelity MMR. Two enzymes are the major determinant of dNTP pools: ribonucleotide reductase (RNR), which converts ribonucleotides into deoxyribonucleotides predominantly during S-phase, and SAMHD1, which degrades dNTPs predominantly outside of S-phase. In Chapters four and five, I quantified antibody hypermutation in B cells lacking SAMHD1 and/or over-expressing RNR. I observed a 2-fold decrease in mutations at A:T bases in cells lacking SAMHD1. This decrease was comparable to the decrease induced by RNR over-expression and was consistent with our hypothesis. Unexpectedly, loss of SAMHD1 also decreased transversion mutations at C:G by about 70%, and almost doubled transition mutations at C:G bases. RNR over-expression had no obvious impact on transversion mutations at C:G, but increased transition mutations at C:G bases similarly to loss of SAMHD1. Furthermore, loss of SAMHD1 decreased AID/BER-dependent antibody class switch recombination, while RNR over-expression did not. These findings indicate that dNTPs play a role in MMR-mediated antibody mutation, as predicted by our hypothesis, but they also indicate a major role for SAMHD1 in AID-induced BER that was not predicted by our hypothesis or by current models of antibody hypermutation. This important finding warrants further investigation to identify the mechanism.
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Turnbull, Charlotte Louise. "Sequence stability of the APC gene : the role of DNA repair mechanisms in colon carcinogenesis." Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/25262.
Повний текст джерелаАнотація:
To study the role of instability in colorectal cancer, this project focused on investigating any inherent sequence-specific mutation frequency of the APC gene. I used an approach where clonal selective advantage imparted by APC mutation is avoided, and have utilised a number of cancer and non-cancer cell lines, and patient tissue samples. As deficient DNA repair has been implicated in a number of CRC conditions, I have also investigated the effect of defective DNA mismatch repair (MMR) and base exclusion repair (BER) on APC mutation frequency and spectrum. In addition, I have also begun to develop a novel technique for directly measuring BER activity in live cells. I show substantial elevation of mutation frequency in the APC gene compared to control gene sequences. This is the first demonstration of inherent sequence instability of APC in normal cells. Polymorphism and evolutionary data indicate that APC is likely to be subject to a high neutral mutation rate and purifying selection. Furthermore, detailed analysis of de novo APC mutations has not implicated the involvement of any alternative mutational mechanisms such as CpG island methylation. Further work is therefore required to understand the sequence instability observed in these normal cells. In the cell lines studied, mutation frequency at all gene sequences was increased in the defective BER background and in heterozygous MMR cells. Detectable APC gene mutation frequency was substantially reduced in cell lines null for MMR. In patient samples a striking effect of Germline MUTYH mutation on APC mutation spectrum was observed in tumour samples, and the consequence of the bi-allelic inactivation of MUTYH on somatic inactivating mutations in APC and K-ras was clear. The findings presented in this thesis provide new understanding of early molecular events during tumourigenesis and how they are affected by defects in DNA repair systems, in particular BER and MMR.
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Bennett, L. "Epigenetic mechanisms involved in the cellular response to DNA damage processed by Base Excision Repair." Thesis, University of Liverpool, 2017. http://livrepository.liverpool.ac.uk/3012159/.
Повний текст джерелаАнотація:
Chromatin remodelling is required for access to occluded sequences of DNA by proteins involved in important biological processes, including DNA replication and transcription. There is an increasing amount of evidence for chromatin remodelling during DNA repair, although this has been mostly focused towards DNA double strand break and nucleotide excision repair. At this time there is little evidence for chromatin remodelling in base excision repair (BER). BER is a highly conserved DNA repair pathway which processes spontaneous endogenous DNA base damages generated by oxidative metabolism, but also those induced by exogenous agents (eg. ionising radiation), to maintain genome stability. The mechanism in which the BER repairs damaged bases has been extensively studied and the repair proteins involved are well known. However in terms of chromatin, BER is poorly understood. It is thought that chromatin remodelling occurs due to accumulating evidence indicating that certain BER enzymes are significantly less efficient at acting on sterically occluded sites and near the nucleosome dyad axis. At this time the mechanisms and enzymes involved to facilitate BER are unknown. Therefore, the study presented in this thesis aimed to identify specific histone modification enzymes and/or chromatin remodellers that are involved in the processing of DNA base damage during BER. A method to generate two mononucleosome substrates with a site specific synthetic AP site (tetrahydrofuran; THF) was used to measure recombinant AP endonuclease 1 (APE1) activity alone, and APE1 in HeLa whole cell extract (WCE) that contain chromatin modifiers. The substrates contained either a THF rotationally positioned in the mononucleosome so the DNA backbone was facing outwards (THF-OUT) so accessible to APE1, or facing inwards (THF-IN) towards the histone octamer and so sterically occluded to APE1. I discovered that the THF-OUT substrate was efficiently processed by recombinant APE1 alone and by APE1 in HeLa WCE. In contrast, recombinant APE1 activity was significantly impeded by THF-IN, but which was efficiently processed by APE1 in HeLa WCE in the presence of factors supporting ubiquitination. This suggested the presence of a chromatin modifier, predictably E3 ubiquitin ligase(s) present in WCE that was increasing THF-IN accessibility to APE1. A sequential chromatography approach was utilised to purify these novel activities from HeLa WCE, and I identified three separate activities capable of stimulating APE1 activity towards the THF-IN mononucleosome. Y-box protein 3 (YBX3) and HECT Domain E3 Ubiquitin Protein Ligase 1 (HECTD1) were identified by mass spectrometry analysis of active fractions and their presence aligned with the APE1 stimulatory activity profile of the THF-IN substrate. Depletion of these proteins using siRNA in HeLa cells decreased cell survival following ionising radiation, and delayed DNA damage repair in both HeLa cells and in normal lung fibroblasts. Together these results suggest that HECTD1 and YBX3 are strong candidates required to facilitate BER through histone ubiquitination and/or chromatin remodelling, and provide new mechanistic information on the process of BER in cellular chromatin.
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Oksenych, Valentyn. "Molecular mechanisms of recruitment of transcription/repair factor TFIIH to the sites of damaged DNA." Strasbourg, 2009. https://publication-theses.unistra.fr/public/theses_doctorat/2009/OKSENYCH_Valentyn_2009.pdf.
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Wnek, Shawn Michael. "Mechanisms of malignant transformation of human urothelial cells by monomethylarsonous acid." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/201495.
Повний текст джерелаАнотація:
Sources of arsenic exposure include air, water, and food from both natural and anthropogenic sources. Arsenic is categorized as a human carcinogen, and is associated with pleiotropic toxicities including cancers of the skin, lung, and bladder. Despite arsenic's long recognition as a human carcinogen, the exact mechanisms of arsenical-induced carcinogenesis are unknown. Arsenic exposure has been shown to cause DNA damage. However, because arsenic does not directly react with DNA, genotoxicity is generally considered to result from indirect mechanisms. The generation of arsenical-induced reactive oxygen species and the inhibition of critical DNA repair systems are believed to contribute to arsenical-induced carcinogenicity. The DNA damaging effects of arsenical exposure and alterations in DNA repair processes were examined within the human bladder urothelial cell line, UROtsa, following continuous exposure to the arsenic metabolite, monomethylarsonous acid [MMA(III)]. Chronic, low-level MMA(III) exposure results in the induction of DNA damage that remains elevated following the removal of MMA(III). Furthermore, data presented herein, defines the critical period in which continuous low-level MMA(III) exposure causes the malignant transformation of the UROtsa cell line. Results indicate that malignant transformation of UROtsa cells is irreversible following 12 wk of low-level MMA(III) exposure. Assessment of the MMA(III)-induced biological alterations leading to the malignant transformation of UROtsa cells following 12 wk of exposure suggest two potential interdependent mechanisms in which MMA(III) may increase the susceptibility of UROtsa cells to genotoxic insult and/or malignant transformation. These mechanisms include MMA(III)-induced DNA damage via the production of reactive oxygen species and the MMA(III)-induced inhibition of poly(ADP-ribose) polymerase-1 as a result of the direct MMA(III)-mediated displacement of zinc.
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Cullen, Jason Kingsley. "Investigating the mechanisms responsible for DNA double-strand break-induced loss of heterozygosity in fission yeast." Thesis, University of Oxford, 2007. http://ora.ox.ac.uk/objects/uuid:95f5dba5-7836-4f63-8c8e-66e6151f615b.
Повний текст джерелаАнотація:
Loss of heterozygosity (LOH) is considered a causal event in the formation of many cancers, with increasing evidence suggesting that DNA double-strand breaks (DSBs) play a major role in its occurrence. Despite its prominence in cancer, however, the precise molecular mechanisms responsible for extensive LOH and how such events are suppressed in normal cells is poorly understood. To investigate the mechanisms responsible for extensive break-induced LOH in eukaryotes, this study took advantage of an assay system in which such events could be identified through screening for loss of an auxotrophic his3+ marker, found ~25kb distal to an HO-endonuclease cut site in a non-essential minichromosome in Schizosaccharomyces pombe. Studies using this system had previously shown that extensive break-induced LOH in wild-type background, whilst infrequent, was predominantly associated with large translocations resulting from both allelic crossovers during G2 phase and breakinduced replication (BIR). Such extensive loss of allele specific information was also found to require rhp55+, rhp51+, rhp54+ and mus81+. This study has identified an additional role for the MRN complex, Rad22 and RPA in such break-induced translocations, suggesting that both allelic crossovers and BIR require homologous recombination (HR) in fission yeast. Surprisingly, break-induced extensive LOH was still observed in HR mutants. In contrast to wild-type cells, however, such extensive LOH was found to arise predominantly through de novo telomere addition at, or near, the break-site. Interestingly, telomere addition was most frequently observed in a rad22Δ background that disrupts HR following end resection. Further analysis demonstrated that de novo telomere addition was also significantly increased in ku70Δ rhp55Δ cells. Moreover, overexpression of rhp51 in rhp55Δ cells led to a substantial reduction in break-induced de novo telomere addition. Together, these findings support a model in which HR prevents de novo telomere addition at DSBs by competing for resected ssDNA ends. In addition to providing information on break-induced LOH this study has identified a requirement for the MRN complex in efficient repair in rhp55Δ cells, which was previously found to occur via sister chromatid recombination (SCR) or a HRdependent end-joining pathway (EJ). Interestingly, deletion of MRN components also resulted in an increase in telomere addition, providing further evidence that HR competes with telomere addition for the repair of DSBs. Overall, these findings shed light on the competitive relationships between pathways of DSB repair/misrepair in S. pombe and how such mechanisms contribute to the prevention or promotion of genome instability.
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Ahrabi, Sara. "Genetic analysis of DNA double-strand break mis-repair mechanisms using the human endogenous HPRT gene." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:2ebfaf6f-cc69-4b1c-918b-9be2280b51ff.
Повний текст джерелаАнотація:
DNA double strand breaks (DSBs) are the most lethal radiation-induced lesions in response to which cells employ either the error free homologous recombination (HR) repair pathway or error prone mechanisms, such as non homologous end joining (NHEJ) and microhomology- mediated end joining (MMEJ). While MMEJ is suppressed by C-NHEJ, the relationship between HR and MMEJ is less clear. In this thesis, I have exploited the human endogenous HPRT gene to develop a novel genetic technology to detect mutation frequencies and signatures in response to DNA DSBs in different genetic backgrounds. Using a sensitive HPRT assay we describe a role for HR genes in suppressing MMEJ in human cells. By monitoring DSB mis-repair, we found that depletion of HR proteins, including RAD51, BRCA2, BRCA1 or SETD2, resulted in a distinct mutational signature associated with significant increases in break-induced mutation frequencies, deletion lengths and the annealing of short regions of microhomology (2 - 6 bp) across the break-site. This signature was dependent on CtIP, MRE11, POLQ and PARP, and thus indicative of MMEJ. In contrast to CtIP or MRE11, depletion of BRCA1 resulted in increased partial resection and MMEJ, thus revealing a functional distinction between these early acting HR factors. Together these findings indicate that HR factors suppress mutagenic MMEJ following DSB resection. Further, I have defined a role for the SETD2 histone methyltransferase in suppressing break-induced mutations, and have shown that CRISPR/Cas9 and ISceI- induced DSBs resulted in different repair profiles.
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Martinez, Alaina R. "Variant requirements for DNA repair proteins in cancer cell lines that use alternative lengthening of telomere mechanisms of elongation." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1479924417740462.
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Saini, Natalie. "Understanding the mechanisms underlying DSB repair-induced mutagenesis at distant loci in yeast." Diss., Georgia Institute of Technology, 2014. http://hdl.handle.net/1853/51843.
Повний текст джерелаАнотація:
Increased mutagenesis is a hallmark of cancers. On the other hand, this can trigger the generation of polymorphisms and lead to evolution. Lately, it has become clear that one of the major sources of increased mutation rates in the genome is chromosomal break formation and repair.
A variety of factors can contribute to the generation of breaks in the genome. A paradoxical source of breaks is the sequence composition of the genomic DNA itself. Eukaryotic and prokaryotic genomes contain sequence motifs capable of adopting secondary structures often found to be potent inducers of double strand breaks culminating into rearrangements. These regions are therefore termed fragile sequence motifs. Here, we demonstrate that in addition to being responsible for triggering chromosomal rearrangements, inverted repeats and GAA/TTC repeats are also potent sources of mutagenesis. Repeat-induced mutagenesis extends up to 8 kb on either side of the break point. Remarkably, error-prone repair of the break by Polζ reconstitutes the repeats making them a long term source of mutagenesis.
Despite its negative connotations for genome stability, the mechanisms underlying the unstable nature of double strand break repair pathways are not known. Previous studies have demonstrated that break induced replication (BIR), a mechanism employed to repair broken chromosomes with only one repairable end, is highly mutagenic, undergoes frequent template switching and often yields half-crossovers. In the work presented here, we show that the instabilities inherent to BIR can be attributed to its unusual mode of synthesis. We determined that BIR proceeds via a migrating bubble with long stretches of single-stranded DNA and culminates with conservative inheritance of the newly synthesized DNA.
We propose that the mechanisms described here might be important for generation of repair-associated mutagenesis in higher organisms. Secondary structure forming repeats like inverted repeats have been found to be enriched in cancer cells. These motifs often constitute chromosomal rearrangement hot-spots and demonstrate the phenomenon of kataegis. This study provides a mechanistic insight into how such breakage-prone motifs contribute to hypermutability of cancer genomes.
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Rivera, Maricruz. "MOLECULAR MECHANISMS OF STRESS RESPONSE IN BRAIN CANCER." Case Western Reserve University School of Graduate Studies / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=case1445956088.
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Colosio, A. "MECHANISMS MEDIATING REPLICATION FORK COLLAPSE AND PROCESSING IN CHECKPOINT DEFECTIVE CELLS." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/234147.
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ABSTRACT
An accurate DNA replication is essential to prevent genome instability events, such as mutations and chromosomal rearrangements that are hallmarks of neoplastic transformation and cancer onset. A dedicated branch of the DNA damage checkpoint maintains the integrity of replicating chromosomes by stabilising replication forks in the presence of genotoxic agents, thus ensuring cell viability. Upon fork collapse, budding yeast checkpoint mutants experiencing replication stress accumulate aberrant replication intermediates, such as gapped and hemireplicated molecules, as well as four-branched structures known as reversed forks. Aberrant replication intermediates are potentially harmful for the cells since they are thought to trigger unscheduled recombination events that cause genome rearrangements. In this PhD thesis, I examined checkpoint-dependent mechanisms controlling fork stability, and I provide in vivo evidence that positive supercoiling accumulating ahead of replication forks is the main mechanical force driving fork reversal. Thus, DNA topology is a critical determinant of replication fork stability in vivo. Furthermore, a 2D-gel screening for enzymatic activities involved in the metabolism of collapsed forks, revealed a novel role for the Sae2 and Dna2 endonucelases in replication intermediates processing.
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Chan, Kara Y. "MECHANISMS OF TRINUCLEOTIDE REPEAT INSTABILITY DURING DNA SYNTHESIS." UKnowledge, 2019. https://uknowledge.uky.edu/toxicology_etds/29.
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Genomic instability, in the form of gene mutations, insertions/deletions, and gene amplifications, is one of the hallmarks in many types of cancers and other inheritable genetic disorders. Trinucleotide repeat (TNR) disorders, such as Huntington’s disease (HD) and Myotonic dystrophy (DM) can be inherited and repeats may be extended through subsequent generations. However, it is not clear how the CAG repeats expand through generations in HD. Two possible repeat expansion mechanisms include: 1) polymerase mediated repeat extension; 2) persistent TNR hairpin structure formation persisting in the genome resulting in expansion after subsequent cell division. Recent in vitro studies suggested that a family A translesion polymerase, polymerase θ (Polθ), was able to synthesize DNA larger than the template DNA. Clinical and in vivo studies showed either overexpression or knock down of Polθ caused poor survival in breast cancer patients and genomic instability. However, the role of Polθ in TNR expansion remains unelucidated. Therefore, we hypothesize that Polθ can directly cause TNR expansion during DNA synthesis. The investigation of the functional properties of Polθ during DNA replication and TNR synthesis will provide insight for the mechanism of TNR expansion through generations.
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Bray, Sian Marian. "Mechanisms and regulation of dsDNA break repair in the Sulfolobus genus of thermophilic archaea." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/290298.
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DNA is constantly subjected to chemical and mechanical damage. The ability to repair the lesions sustained is essential for all life. Double stranded DNA (dsDNA) breaks are especially toxic as both antiparallel strands of DNA are severed. The most high fidelity mechanism available to repair this damage is homologous recombination, a mechanism that uses homology from the sister chromatid to replace any lost information. Key proteins involved in maintaining genomic stability this way are conserved in all domains of life. One such component is the Mre11/Rad50 complex that is involved in the initial recognition of damage and recruitment of subsequent repair factors. Understanding the function of this DNA repair complex and any associated proteins has implications for human cancers and aging. The proteins of thermophilic archaea present an excellent opportunity to study these systems in a robust, tractable and eukaryote-like system. Archaea are in many ways biochemically unique, for example they are the only domain capable of methanogenesis. However archaea share a high level of homology with eukaryotes in many essential cellular processes such as DNA replication, homologous recombination and protein degradation. In thermophilic archaea the mre11/rad50 genes are clustered in an operon with the herA/nurA genes that form a helicase/nuclease complex. This has lead to speculation that the four proteins work together during homologous recombination to produce the 3' overhangs required by RadA to identify homology. As part of this investigation I have performed extensive bioinformatic searches of a variety of archaeal/bacterial systems. These analyses have revealed operonic linkages to other known recombinational helicase/nucleases, such as AddAB and RecBCD. These genomic linkages are especially prevalent in thermophilic organisms suggesting their functional relevance is particularly acute in organisms exposed to a high amount of genomic stress. Comparison of the evolutionary trees, constructed for each protein, makes a single genomic linkage event the most likely scenario, but cannot definitively exclude other possibilities. Exhaustive attempts were made to demonstrate an interaction between Mre11/Rad50 and HerA/NurA. Despite analysis by nickel/cobalt pulldown, immunoprecipitation, analytical gel filtration, ITC and OCTET an interaction could not be confirmed or definitively dismissed. However in the process an interesting Rad50 tetrameric assembly was identified and attempts were made to crystalize it. Hexameric helicases and translocases are key to the replication and DNA packaging of all cellular life and multiple viruses. The hexameric translocase HerA is a robust model for investigating the common features of multimeric ATPases as it is extremely stable and experimentally tractable. Here it is revealed that HerA exists in a dynamic equilibrium fluctuating between hexameric and heptameric forms with rapidly interchanging subunits. This equilibrium can be shifted to heptamer by buffering conditions or towards the hexamer by the physical interaction with the partnering nuclease NurA, raising the possibility that these alternate states may play a role in translocase assembly or function. A novel C-terminal brace, (revealed by a collaborative crystallographic structure) is investigated; as well as stabilizing the assembly, this brace reaches over the ATPase active site of its neighbouring subunit. It is seemingly involved in the conversion of energy generated by ATP hydrolysis into physical movement in the central channel of the hexamer. The regulation of homologous recombination is extremely important to prevent aberrant activity, resulting in mutations and genome reorganization. In eukaryotic organisms, it is well established that post-translational modifications and protein turnover at the proteosome play important roles in this control. In particular, there is significant interest currently in the ubiquination-proteasome destruction pathway as a mechanism for extracting DNA repair components from chromatin at the termination of the DNA repair process. To date no Ubiquitin proteins have been identified in the Archaea, however related proteins URMs/SAMPs (Ubiquitin Related Modifier/Small Archaeal Modifier Protein) have previously been identified. URMs are thought to have evolved from a common antecedent to eukaryotic ubiquitin and likely represent an evolutionary 'missing link' in the adaption of sulphur transfer proteins for covalent modifications. There has been speculation that Urm1 may play a similar role to ubiquitin in the proteasome degradation pathway and we have recently provided evidence to corroborate this. Here the potential for modification of Mre11/Rad50/HerA/NurA by Urm1 was investigated. Indeed Rad50 shows evidence of clear urmylation both in vivo and in vitro. Western blotting and mass spec analysis confirmed the covalent attachment of Urm1 to Rad50. Furthermore I present preliminary evidence that this urmylation can lead to the destruction of Rad50 via a direct physical interaction with the proteasome. This is the first evidence of such a regulatory system for Rad50. Investigating the urmylation and destruction of Rad50 was closely linked to investigating the archaeal proteasome, a close homologue of the eukaryotic proteasome. To date the majority of archaeal core proteasomes examined were believed to consist of only two subunits; alpha and beta. The subunits are arranged into heptameric rings, which then form an alpha/beta/beta/alpha stack with a single channel running through the centre of all four rings. Here we reveal that in Sulfolobus species the inner catalytic chambers are made up of mixed beta rings composed of two subunits. The first plays a crucial structural role but appears catalytically inert, while the second conveys catalytic activity. Here we investigate an inactive complex, containing only the structural beta subunit, and an active complex, containing both beta subunits. First, electron microscopy was performed on both complexes revealing the expected four-layered toroidal stack. Both complexes were subsequently investigated crystallographically. A 3.8 Å structure was determined for the inactive complex. As well as being one of the few archaeal core proteasome structures, this is also an important first step towards structurally investigating the novel three-subunit proteasome. The discovery of active and inactive beta subunits in the archaea brings them even closer to eukaryotic proteasomal systems, making the archaea even more valuable as model systems.
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Bradburn, A. K. "Effect of DNA damage and repair mechanisms in human haematopoiesis and their role in chemoresistance of acute myeloid leukaemia." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/1543334/.
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Most standard AML chemotherapy regimes utilize Ara-C, a cytotoxic compound inflicting DNA damage in proliferating cells and inducing cell death. Comparing normal and malignant haematopoietic cells in their response to Ara-C will determine whether differing responses to DNA damage and activation of DNA repair mechanisms contribute to chemoresistance. Moreover, DNA repair in primitive human haematopoiesis and LIC’s is still not well established. Using AML cell lines, cord blood and CD34+ AML patient samples, a characterization of the DNA damage response post Ara-C treatment was performed. AML cell lines were shown to have a range of sensitivities with significant increases in DNA damage after Ara-C exposure. However, cell lines resistant to Ara-C, were quicker to recover their DNA damage, with a faster revival of cell numbers and reduction in apoptosis. Similar findings were also observed when using ionizing radiation as a source of DNA damage. Analysis of the DNA repair mechanisms non-homologous end joining (NHEJ) and homologous recombination (HR) by 53BP1 and RAD51 localization respectively, showed NHEJ was preferentially used in response to Ara-C, with low rates of HR. Normal haematopoietic stem and progenitor populations were compared with leukaemia initiating and tumour populations of CD34+ AML patient samples in vivo. Normal stem populations showed significant increases in apoptosis with enrichment in cells in G0 after exposure to Ara-C. Progenitor cells were more refractory to treatment and displayed a G1/S-phase accumulation. When observing AML patient samples it appeared they either behaved in a progenitor, or a stem like manner, showing heterogeneous responses overall. Just like the AML cell lines NHEJ was primarily observed, with some basal level of activation. Overall there are differences in normal stem progenitor and cells in response to Ara-C, with AML patient samples displaying a more heterogeneous response to DNA damage.
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Haapa-Paananen, Saija. "The mechanisms, applications, and target site selection of bacteriophage Mu minimal in vitro DNA transposition reaction." Helsinki : University of Helsinki, 2002. http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/haapa-paananen/.
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Duncan, Hal Fergus. "Epigenetic approaches : the emerging role of histone deacetylase inhibitors (HDACis) in promoting dental pulp cell repair mechanisms in vitro." Thesis, University of Birmingham, 2016. http://etheses.bham.ac.uk//id/eprint/6975/.
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Despite recent improvements in the clinical outcomes of vital pulp treatment, existing approaches remain non-specific and unpredictable. Developing biologically-based therapies that promote pulp regeneration is critical. Epigenetic modifications of DNA and histones control cellular processes, including proliferation, mineralisation and stem cell fate, and therefore offer exciting therapeutic opportunities. Chromatin acetylation can be altered pharmacologically using histone-deacetylase-inhibitors (HDACis), which relax its structure and modulate transcription. This project investigated regenerative-associated HDACi effects in vitro on a cell-line and primary dental-pulp-cells (DPCs), using proliferation, viability, mineralisation, cell-migration, enzyme activity, high-throughput gene/protein expression and pathway analyses. HDACis increased DPC differentiation and mineralisation-associated gene/protein expression at concentrations, which did not reduce viability. Primary DPC mineralisation was promoted without altering cell viability/apoptosis, indicating a resistance to HDACi-mediated toxicity compared with cell-lines. HDACi-induced DPC reparative processes were mediated by matrix metalloproteinase (MMP) expression and activity. MMP-13 inhibition further increased mineralisation-associated events, but decreased cell-migration indicating a novel role for MMP-13 in pulpal repair. HDACi solutions released a range of previously characterised and unreported bioactive dentine matrix components, which may further supplement regenerative capability in vivo. Results demonstrate that HDACi directly stimulate DPC repair-associated events, highlighting their potential for augmenting dental materials or pulp-engineering scaffolds for regenerative endodontics.
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Makhalova, Julia. "Molecular mechanisms of cisplatin induced neutrotoxicity formation and repair of specific DNA lesions in different cell types of nervous tissue /." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971010064.
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Shell, Steven Michael. "Structural and Biochemical Investigation of the Molecular Mechanisms of DNA Response and Repair in Humans and Escherichia coli." Digital Commons @ East Tennessee State University, 2008. https://dc.etsu.edu/etd/1937.
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The genomes of all living cells are under constant attack from both endogenous and exogenous agents that damage DNA. In order to maintain genetic integrity a variety of response pathways have evolved to recognize and eliminate DNA damage. Replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, is a required factor for all major DNA metabolisms. Although much work has been done to elucidate the nature of the interaction between RPA and ssDNA currently there is no structural information on how the full-length protein binds to ssDNA. This study presents a novel examination of the full nucleoprotein complex formed between RPA and ssDNA. We identified three previously unknown contacts between ssDNA and lysine residues in DNA binding domain C located in the p70 subunit. This represents the first single amino-acid resolution determination of how full-length RPA contacts ssDNA. The Ataxia-Telangiectasia Mutated and RAD3-Related (ATR) mediated DNA damage checkpoint and nucleotide excision repair (NER) pathway are primarily responsible for repair of UV-C-induced photolesions in DNA. However, it is unclear how these two pathways are coordinated. We found the ATR-dependent checkpoint induces a rapid nuclear accumulation of the required NER factor Xeroderma pigmentosum group A (XPA) in both a dose- and time-dependent fashion. Also, using surface topology mapping we have defined an α-helix motif on XPA required for XPA-ATR complex formation necessary for XPA phosphorylation. In addition, we have determined that XPA phosphorylation promotes repair of persistent DNA lesions, such as cyclobutane pyrimidine dimers. The basis for initial damage recognition in NER is structural distortion of duplex DNA; however, the effects of adduct structure and sequence on strand opening and recognition are unclear. Using the E. coli NER system we determined that the identity of the adduct dictates the size of the strand opening generated by the UvrA2B complex. In addition we found that the sequence immediately surrounding the damaged nucleotide affects damage recognition by influencing the amount of helical distortion induced by the adduct. These effects are a result of the equilibrium conformation the adduct adopts in addition to the amount of hydrogen bonding available to maintain the structure.
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Koskiniemi, Sanna. "Dynamics of the bacterial genome rates and mechanisms of mutation /." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-111428.
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Shimabukuro, Fernanda. "Avaliação in vitro da cisplatina, em linfócitos de pacientes com melanoma cutâneo, por meio de testes citogenéticos." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/5/5160/tde-03092010-111828/.
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O melanoma cutâneo maligno é uma lesão neoplásica originada nos melanócitos epidérmicos, sendo altamente invasiva e agressiva, com elevada taxa de mortalidade, cuja incidência vem aumentando nos últimos anos. O tratamento do melanoma é cirúrgico e os pacientes com metástase podem receber quimioterapia com cisplatina que ao formarem adutos com o DNA alteram o processo de replicação da célula cancerosa. Sugere-se que os sistemas de reparo do DNA tenham um papel importante na etiologia do melanoma (reparo deficiente) e no tratamento do mesmo (eficiente eliminação dos adutos). A identificação prévia da resposta dos pacientes com melanoma ao tratamento com cisplatina pode ser um indicador biológico importante na clínica oncológica. O presente trabalho teve como objetivo, a partir de linfócitos de sangue periférico de pacientes com melanoma e de controles, avaliar o dano no DNA antes e após a adição, in vitro, de cisplatina (10?M, 100?M e 250?M), além de estimar a capacidade de reparo do DNA, após a retirada da droga (1h, 2,5h e 5h). Foram utilizados os testes do micronúcleo (MN - dano basal) e do Cometa (dano basal, ação da cisplatina e reparo do DNA). A análise citogenética foi possível em 20 pacientes com melanoma (10 homens e 10 mulheres, média de 50,6 ± 5,9 anos) e 19 controles (9 homens e 10 mulheres, média de 49,9 ± 5,5 anos) que também responderam a um questionário sobre hábitos e tipos de exposição a fatores de risco ao melanoma. A frequência do dano basal pelo teste do MN e do Cometa em linfócitos de pacientes (MN = 1,2 ± 1,2 e Cometa = 59,3 ± 62,5) foi praticamente o dobro da observada nos controles (MN = 0,6 ± 1,0 e Cometa = 35,3 ± 18,6) embora a diferença entre os grupos, em ambos os testes, não tenha sido considerada estatisticamente significante (p=0,23 e p=0,85, respectivamente). O tratamento in vitro com cisplatina, em comparação com o dano basal, aumentou a frequência de Cometas nas três concentrações estudadas (10?M, 100?M e 250?M) tanto para os pacientes (65,50 ± 50,06, 72,74 ± 50,89 e 77,26 ± 44,16) quanto para os controles (66,53 ± 49,85, 66,53 ± 26,33 e 81,74 ± 43,12) diferença esta considerada significante somente para o grupo controle, nas três concentrações avaliadas (p=0,0175, p=0,0002, e p=0,0002, respectivamente). Quanto aos diferentes tempos de reparo (1h, 2,5h e 5h), após a retirada de cisplatina nas diferentes concentrações estudadas, verificou-se aumento na frequência média de Cometas tanto para os pacientes com melanoma (93,88 ± 33,7, 101,75 ± 35,7 e 99,31 ± 32,30) quanto para os controles (92,45 ± 38,4, 100,82± 38,8 e 100,81± 31,7), diferença que foi estatisticamente significante quando comparada ao dano basal observado nos pacientes (p<0,001) e nos controles (p<0,001). Resultados semelhantes foram observados quando comparados em conjunto os escores dos tempos de reparo com o escore obtido após tratamento com cisplatina nos pacientes (71,09 ± 48,2; p<=0,005) e nos controles (71,59 ± 40,5; p<=0,005). Os resultados obtidos parecem indicar um padrão de resposta semelhante em relação à cisplatina e ao reparo do DNA nos dois grupos de indivíduos avaliados. O período de incubação das células, após a retirada da cisplatina, bem como o número de indivíduos avaliados podem ter influenciado nos resultados obtidos. Por outro lado, a resposta observada nos linfócitos in vitro, pode não ser representativa do efeito in vivo da célula tumoral. Entretanto, a identificação de marcadores de resposta a tratamentos com quimioterápicos, a partir de linfócitos de sangue periférico pode ser uma estratégia de pesquisa importante na prática clínica, inclusive para o melanoma.Cutaneous melanoma is a malignant tumor originated from epidermal melanocytes, highly invasive and aggressive, with high mortality, and incidence that has been increasing over the years. The treatment for melanoma is surgery and patients with metastasis may receive chemotherapy with cisplatin, that results in DNA adducts that alters the replication process in cancer cells. It is suggested that the DNA repair systems have an important role in the etiology of melanoma (risk due to deficient repair) and treatment efficiency (removal of DNA adducts can decrease the treatment results). The prior identification of the response of melanoma patients to treatment with cisplatin may be an important biological marker in clinical oncology. The aim of this study was to assess, in peripheral blood lymphocytes from melanoma patients and controls, the DNA damage before and after the addition of cisplatin (10?M, 100?M and 250?M), in vitro, and estimate the capacity of DNA repair after drug removal (1h, 2.5h and 5h). The micronucleus test (MN - basal DNA damage) and the Comet assay (basal DNA damage, action of cisplatin and DNA repair) were used for the evaluation. Cytogenetic analysis was performed in 20 melanoma patients (10 men and 10 women, average age 50.6 ± 5.9 years old) and 19 controls (9 men and 10 women, average age 49.9 ± 5.5 years old) who also answered a questionnaire on habits and types of exposure to risk factors for melanoma. The frequency of basal DNA damage by the MN test and the Comet assay in lymphocytes from patients (MN = 1.2 ± 1.2 and Comet = 59.3 ± 62.5) was nearly twice the observed in controls (MN = 0, 6 ± 1.0 and Comet = 35.3 ± 18.6), although the difference between the groups in both tests was not considered statistically significant (p = 0.23 and p = 0.85, respectively). The in vitro treatment with cisplatin, compared with the basal DNA damage, increased the frequency of Comets in the three studied concentrations (10?M, 100?M and 250?M) for patients (65.50 ± 50.06, 72.74 ± 50.89 and 77.26 ± 44.16) and for the controls (66.53 ± 49.85, 66.53 ± 26.33 and 81.74 ± 43.12) and the difference was statistically significant only for the control group, for all cisplatin concentrations (p = 0.0175, p = 0.0002 and p = 0.0002, respectively). Considering the different repair times (1h, 2.5h and 5h), after removal of cisplatin at different concentrations, there was an increase in the mean frequency of Comets for both melanoma patients (93.88 ± 33.7, 101.75 ± 35.7 and 99.31 ± 32.30) and for the controls (92.45 ± 38.4, 100.82 ± 38.8 and 100.81 ± 31.7), and the difference was statistically significant when the repair Comet score was compared to the basal DNA damage observed in patients (p <0.001) and controls (p <0.001). Similar results were observed when the Comet scores of repair times were compared to the Comet scores obtained after treatment with cisplatin in patients (71.09 ± 48.2, p <= 0.005) and controls (71.59 ± 40.5, p <= 0.005). The results seem to indicate a similar pattern of response to cisplatin and DNA repair in both groups of subjects evaluated. The period of incubation of the cells after cisplatin removal and the number of individuals studied may have influenced the results. The lymphocytes\' response, in vitro, to cisplatin may not be representative of the in vivo effect of tumor cell. However, the identification of markers of response to treatment with chemotherapy from peripheral blood lymphocytes may be an important research strategy in clinical practice, including melanoma.