Dissertations / Theses on the topic 'DNA Single-strand Repair Pathway'

Kyoto University (京都大学)
0048
新制・課程博士
博士(人間・環境学)
甲第19068号
人博第721号
新制||人||173(附属図書館)
26||人博||721(吉田南総合図書館)
32019
京都大学大学院人間・環境学研究科相関環境学専攻
(主査)教授 小松 賢志, 教授 宮下 英明, 准教授 三浦 智行
学位規則第4条第1項該当

Genetic instability is the driver of cancer initiation and progression. The human single stranded DNA binding protein 2 is known to be involved in the prevention of such genetic instability. This project has highlighted how human single stranded DNA binding protein 2 is involved in a particular DNA repair pathway called, base excision repair. The researcher identified the role of human single stranded DNA binding protein 2 in the removal of uracils that have been added by mistake to the human genome. This project details the mechanism by which uracils are removed from the genome, shedding light on the evolution of the cancer genome and the mechanism through which genetic stability can occur.

The external environment and internal cellular processes generate DNA double strand breaks (DSBs), a particularly toxic form of DNA damage that can result in chromosome fragmentation, replication failure, mutagenesis and cell death. Cells have evolved effective mechanisms to preserve the mtegrity of the genome including DNA damage signalling, cell cycle checkpoint activation and DNA repair.

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/NBS1
DNA 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

XRCC1 plays a major role in the repair of these lesions in mammalian cells by binding and/or activating many components of the single-strand break repair (SSBR) pathway. One such component is polynucleotide kinase (PNK) which possesses a divergent forkhead associated (FHA) domain that binds CK2-phosphorylated XRCC1. Aprataxin has a similar divergent FHA domain that also interacts with XRCC1 and which has been implicated in SSBR. In this thesis, yeast two-hybrid analysis indicated that PNK interacted with the pro-apoptotic protein Hippi in a manner dependent on the PNK FHA domain. In addition, a novel protein containing a similar FHA domain to PNK and aprataxin was identified and denoted APLF (Aprataxin and PNK-Like Factor). APLF was also shown to bind XRCC1 in a manner dependent on its FHA domain. Furthermore, this interaction was greatly stimulated by CK2-phosphorylation of XRCC1. APLF interacted with the double-strand break repair (DSBR) factor XRCC4. APLF was modified following DNA damage, presumably by phosphorylation. Nuclear localisation of YFP-APLF was promoted by the presence of XRCC1. Moreover, YFP-APLF colocalised with RFP-XRCC1 in DNA damage-induced nuclear foci following H₂O₂ treatment. Novel interaction partners of APLF identified by employing a yeast two-hybrid library screen included Ku86/XRCC5 and KEAP1. These data suggest a role for APLF in the cellular response to DNA strand breaks.

Single-strand breaks (SSBs) are one of the most common types of lesion arising within cells; formed by attack of genotoxic agents on the DNA, as well as enzymatically during normal cellular processes. Although the single-strand break repair (SSBR) pathway is relatively well characterised, and many components have been extensively studied in vitro, little is known of how this pathway operates in vivo when DNA is complexed with histone proteins to form chromatin. This compaction of the DNA into nucleosomal structures has the potential to inhibit repair, by sterically blocking access of repair factors to sites of DNA damage. Whilst previous studies have shown that repair of DNA double-strand breaks and UV-induced lesions are associated with alterations in chromatin structure, through covalent modification of histone proteins and nucleosome remodeling, few similar observations have been made concerning SSBR. Here, I have produced and employed mammalian cell lines stably expressing fluorescently-tagged histone proteins to analyse the dynamics of chromatin occurring upon DNA damage. Localised damage was introduced using micro-irradiation with a UV-A laser, and the histone proteins at the site of damage visualized in real-time using confocal microscopy. Through this method, I have identified a rearrangement of chromatin structure in the vicinity of DNA strand breaks in mammalian cells, resulting in a mobilization of histone proteins at the site of damage. Furthermore, I have shown that this alteration is partially dependent on the activities of both the SSBR factor poly(ADP-ribose) polymerase 1 (PARP-1), and the phosphoinositide 3-kinase-like kinase (PIKK) Ataxia telangiectasia mutated (ATM). I have examined a potential requirement for ATM in SSBR, and found no evidence of this, suggesting that the effects of PARP-1 and ATM on histone mobilization are reflective of the independent contributions of repair of single- and double-strand breaks respectively.

Aprataxin protects nuclear and mitochondrial DNA against genotoxic stress, and loss-of-function mutations in the APTX gene cause the autosomal recessive cerebellar ataxia, Ataxia Oculomotor Apraxia 1 (AOA1) in humans. In an effort to extend current understanding of aprataxin function, this thesis examines the roles of aprataxin, especially in response to oxidative damage. Firstly, involvement of aprataxin during the gap-filling as well as the end-processing steps of single strand break repair were demonstrated using an in vitro single strand break repair assay using synthetic DNA substrates, cell-free lysates and/or recombinant proteins. Next, loss-of-function studies were conducted in Aptx-/- mouse embryonic fibroblasts (MEFs) and tissues from adult mice harbouring a toxic gain-of-function mutant form of superoxide dismutase1 (SOD1G93A). Expression of the mutant SOD1G93A enhanced sensitivity to oxidative damage in aprataxin-deleted cells and revealed an accelerated senescence and attenuated somatic growth phenotype. Together these findings suggest that aprataxin function is involved in optimal repair of single strand breaks and is therefore critical in maintaining cell function in situations of elevated oxidative stress.

En réponse aux dommages de son génome, le choix par la cellule de la voie de réparation de l'ADN est un crucial par ses conséquences en termes de mutagénèse et de survie. Pour faire face aux cassures double-brin de l'ADN (CDB), les cellules humaines possèdent deux voies principales qui consistent soit à rejoindre les extrémités de la cassure par jonction d'extrémités non-homologues (voie conventionnelle C-NHEJ), soit à reconstituer par recombinaison homologue la séquence clivée en copiant son double non endommagé présent après la réplication (voie RH). La RH nécessite de dégrader l'un des brins d'ADN de part et d'autre de la cassure. Cette dégradation produit de courts fragments d'ADN simple-brin, connus pour aider à signaler le dommage à la cellule. Dans ce travail, nous avons évalué directement l'effet de ces fragments d'ADN simple brin sur la réparation des CDB dans des expériences biochimiques et cellulaires. Nous montrons que de courts fragments d'ADN simple-brin inhibent la C-NHEJ en inactivant sa protéine clef Ku, tout en stimulant une forme minoritaire de jonction des cassures dite NHEJ alternative (A-EJ). Ces travaux permettent de mieux comprendre comment la réparation par la voie peu connue A-EJ peut s'exprimer dans les cellules mais aussi d'envisager des stratégies pour piloter la réponse des cellules cancéreuses aux thérapies induisant des CDB
In response to DNA damage, the choice made by the cells between DNA repair mechanisms is crucial for mutagenic and survival outcomes. In humans, DNA double-strand breaks are repaired by two mutually-exclusive mechanisms, homologous recombination or end-joining. Among end-joining mechanisms, the main process is classical non-homologous end-joining (C-NHEJ) which relies on Ku binding to DNA ends and DNA Ligase IV (Lig4)-mediated ligation. Mostly under Ku- or Lig4-defective conditions, an alternative end-joining process (A-EJ) can operate and exhibits a trend toward microhomology usage at the break junction. Homologous recombination relies on an initial MRN-dependent nucleolytic degradation of one strand at DNA ends. This process, named DNA resection generates 3' single-stranded tails necessary for homologous pairing with the sister chromatid. While it is believed from the current literature that the balance between joining and recombination processes at DSBs ends is mainly dependent on the initiation of resection, it has also been shown that MRN activity can generate short single-stranded DNA oligonucleotides (ssO) that may also be implicated in repair regulation. In this work, we evaluate the effect of ssO on end-joining at DSB sites both in vitro and in cells. Under both conditions, we report that ssO inhibit C-NHEJ through binding to Ku and favor repair by the Lig4-independent microhomology-mediated A-EJ process. Our data bring new clues in the understanding of the cellular response to DNA double-strand breaks

The goal of my research was to obtain a quantitative understanding of the mechanisms of DNA double-strand break (DSB) repair, and the activation of the tumor suppressor p53 in response to DSBs in human cells. In Chapter 2, we investigated how the kinetics of repair, and the balance between the alternate DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), change with cell cycle progression. We developed fluorescent reporters to quantify DSBs, HR and cell cycle phase in individual, living cells. We show that the rates of DSB repair depend on the cell cycle stage at the time of damage. We find that NHEJ is the dominant repair mechanism in G1 and in G2 cells even in the presence of a functional HR pathway. S and G2 cells use both NHEJ and HR, and higher use of HR strongly correlates with slower repair. Further, we demonstrate that the balance between NHEJ and HR changes gradually with cell cycle progression, with a maximal use of HR at the peak of active replication in mid-S. Our results establish that the presence of a sister chromatid does not affect the use of HR in human cells. Chapter 3 examines the sensitivity of the p53 pathway to DNA DSBs. We combined our fluorescent reporter for DSBs with a fluorescent reporter for p53, to quantify the level of damage and p53 activation in single cells. We find that the probability of inducing a p53 pulse increases linearly with the amount of damage. However, cancer cells do not have a distinct threshold of DSBs above which they uniformly induce p53 accumulation. We demonstrate that the decision to activate p53 is potentially controlled by cell-specific factors. Finally, we establish that the rates of DSB repair do not affect the decision to activate p53 or the dynamical properties of the p53 pulse. Collectively, this work emphasizes the importance of collecting quantitative dynamic information in single cells in order to gain a comprehensive understanding of how different DNA damage response pathways function in a coordinated manner to maintain genomic integrity.

La réparation des coupures de double brin (DSB) de l'ADN par une jonction d'extrémité non homologue (NHEJ) nécessite de multiples protéines pour reconnaître et lier les extrémités de l'ADN, les traiter pour des raisons de compatibilité et les lier ensemble. Nous avons construit de nouveaux substrats d'ADN pour la nano-manipulation à une seule molécule nous permettant de détecter, de sonder et de rompre mécaniquement la synapsis du DSB en temps réel par des composants spécifiques du NHEJ humain. DNA-PKcs et Ku permettent la synapsis des extrémités de l'ADN à une échelle de temps inférieure à la seconde, et l'ajout de PAXX étend cette durée de vie à ~ 2 secondes. Une addition supplémentaire de XRCC4, XLF et Ligase IV a entraîné une synapsis à l'échelle minute et conduit à une réparation robuste des deux brins de l'ADN nanomanipulé. Contrairement à PAXX, un long ARN non codant LINP1 peut également aider la DNA-PK à attacher ensemble les extrémités de l'ADN, ce qui pourrait être plus nécessaire lorsque les extrémités de l'ADN se séparent. De plus, nous interrogeons également le système bactérien Bacillus subtilis NHEJ, qui vient de composer Ku et la ligase D, la bactérie Ku peut également lier l’ADN et introduire la Ligase D renforcer la synapsis et conduire à la ligature. La contribution énergétique des différents composants à la stabilité synaptique est généralement faible, à l’échelle de quelques kCal / mol. Nos résultats combinés définissent les règles d’assemblage des machines NHEJ et révèlent l’importance des interactions faibles, rapidement rompues même sous des forces sous-picoNewton, dans la régulation de ce système chimico-mécanique à plusieurs composants pour l’intégrité du génome. De plus, nous identifions également un nouveau modèle de clivage à double brin d’ADN régulé par Cas9 PAM. En résumé, ce travail de thèse porte sur la génération DSB et le processus détaillé allant de la connexion de l’ADN à la ligature
Repairing DNA double-strand breaks (DSBs) by non-homologous end-joining (NHEJ) requires multiple proteins to recognize and bind DNA ends, process them for compatibility, and ligate them together. We constructed novel DNA substrates for single-molecule nano-manipulation allowing us to mechanically detect, probe, and rupture in real-time DSB synapsis by specific human NHEJ components. DNA-PKcs and Ku allow DNA end synapsis on the sub second timescale, and addition of PAXX extends this lifetime to ~2s. Further addition of XRCC4, XLF and Ligase IV resulted in minute-scale synapsis and led to robust repair of both strands of the nanomanipulated DNA. In contrast with PAXX, a long non-coding RNA LINP1 can also help DNA-PK to tether DNA ends together, which could be more required when DNA ends falling apart in distance. Moreover, we also interrogate the bacteria Bacillus subtilis NHEJ system, which just composed Ku and Ligase D, bacteria Ku also can tether DNA together and introducing the Ligase D strengthen the synapsis and lead to ligation. The energetic contribution of the different components to synaptic stability is typically small, on the scale of a few kCal/mol. Our combined results define assembly rules for NHEJ machinery and unveil the importance of weak interactions, rapidly ruptured even at sub-picoNewton forces, in regulating this multicomponent chemomechanical system for genome integrity. Moreover, we also identify a novel the DNA double strand cleavage pattern regulated by Cas9 PAM. In sum, this PhD work investigates the DSB generation and the detailed process from DNA end tethering to ligation

La réparation des cassures double brin (CDB) de l'ADN par recombinaison est essentielle au maintien de l'intégrité du génome de tous les être vivants. Ce processus doit cependant être finement régulé puisque la recombinaison peut générer des mutations ou des réarrangements chromosomiques, parfois extrêmement délétères pour la cellule. Les CDB peuvent être réparées par deux mécanismes : la recombinaison non homologue (ou jonction des extrémités d'ADN) ou la recombinaison homologue (impliquant une homologie de séquence entre les molécules recombinantes). Dans les cellules somatiques, les deux voies principales de recombinaison homologue (RH) sont la voie Synthesis Dependent Strand Annealing (SDSA) dépendante de la recombinase RAD51 et la voie Single Strand Annealing (SSA) indépendante de RAD51. Nos résultats ont d'abord mis en évidence un rôle inattendu de XRCC2, RAD51B et RAD51D - trois paralogues de RAD51 - dans la voie SSA. Nous avons confirmé que la fonction de la protéine XRCC2 dans la voie SSA ne dépend pas de RAD51, ce qui démontre que certains paralogues de RAD51 ont acquis des fonctions indépendantes de la recombinase. La différence de sévérité des phénotypes des mutants individuels ainsi que les analyses d'épistasie menées sur le double et le triple mutant suggèrent des fonctions individuelles de ces protéines au cours du SSA. Nous proposons qu'elles facilitent l'étape d'hybridation des deux séquences complémentaires situées de part et d'autre de la cassure, bien que ceci reste à confirmer par des études in vitro. L'étude des fonctions de l'hétérodimère XPF-ERCC1 - un complexe impliqué dans le clivage des extrémités d'ADN non homologues au cours des voies de RH - a révélé un rôle inhibiteur de ce complexe sur la voie SDSA. Cette action est dépendante de son activité endonucléasique et serait liée au clivage des longues extrémités 3' sortantes réalisant l'invasion d'un duplex d'ADN homologue, l'étape initiale de la voie SDSA. Notre étude a de plus confirmé que le rôle du complexe dépend de la longueur des extrémités non homologues chez Arabidopsis, comme chez les mammifères et la levure. Bien que le complexe XPF-ERCC1 soit essentiel au clivage des longues extrémités d'ADN non homologue, il n'est pas requis à l'élimination des courtes extrémités au cours de la RH
The repair of DNA double-strand breaks (DSB) by recombination is essential for the maintenance of genome integrity of all living organisms. However, recombination must be finely regulated as it can generate mutations or chromosomal rearrangements, sometimes extremely deleterious to the cell. DSB can be repaired by two classes of recombination mechanism: non-homologous recombination (or DNA End Joining) or homologous recombination (implicating DNA sequence homology between the recombining molecules). In somatic cells, the two main pathways of homologous recombination (HR) are RAD51-dependent Synthesis Dependent Strand Annealing (SDSA) and RAD51-independent Single Strand Annealing (SSA). Our results have demonstrated an unexpected role of XRCC2, RAD51B and RAD51D - three RAD51 paralogues – in the SSA pathway. We confirmed that the function of XRCC2 in SSA does not depend upon RAD51, thus demonstrating that some RAD51 paralogues have acquired RAD51 recombinase-independent functions. The different severities of individual mutant phenotypes and epistasis analyses carried out on the double and triple mutants suggest individual functions of these proteins in SSA recombination. We propose that they facilitate hybridization of the two complementary sequences located on both sides of the break, although this remains to be confirmed by in vitro experiments. Study of the roles of XPF-ERCC1 - a complex involved in the cleavage of non-homologous DNA ends during HR - revealed an inhibitory role of this complex on the SDSA pathway. This is dependent on its endonuclease activity and is probably due to the cleavage of long 3' ends performing the homologous DNA duplex invasion, the initial step of the SDSA pathway. Our analyses also confirmed that the role of the complex depends on the length of the nonhomologous ends, as seen in mammals and yeasts. Although XPF-ERCC1 is essential for the cleavage of long nonhomologous DNA ends, it is not required for the elimination of short ends during HR

Neurodegenerative disorders are heterogenous in nature and include a range of ataxias with oculomotor apraxia, which are characterised by a wide variety of neurological and ophthalmological features. This family includes recessive and dominant disorders. A subfamily of autosomal recessive cerebellar ataxias are characterised by defects in the cellular response to DNA damage. These include the well characterised disorders Ataxia-Telangiectasia (A-T) and Ataxia-Telangiectasia Like Disorder (A-TLD) as well as the recently identified diseases Spinocerebellar ataxia with axonal neuropathy Type 1 (SCAN1), Ataxia with Oculomotor Apraxia Type 2 (AOA2), as well as the subject of this thesis, Ataxia with Oculomotor Apraxia Type 1 (AOA1). AOA1 is caused by mutations in the APTX gene, which is located at chromosomal locus 9p13. This gene codes for the 342 amino acid protein Aprataxin. Mutations in APTX cause destabilization of Aprataxin, thus AOA1 is a result of Aprataxin deficiency. Aprataxin has three functional domains, an N-terminal Forkhead Associated (FHA) phosphoprotein interaction domain, a central Histidine Triad (HIT) nucleotide hydrolase domain and a C-terminal C2H2 zinc finger. Aprataxins FHA domain has homology to FHA domain of the DNA repair protein 5’ polynucleotide kinase 3’ phosphatase (PNKP). PNKP interacts with a range of DNA repair proteins via its FHA domain and plays a critical role in processing damaged DNA termini. The presence of this domain with a nucleotide hydrolase domain and a DNA binding motif implicated that Aprataxin may be involved in DNA repair and that AOA1 may be caused by a DNA repair deficit. This was substantiated by the interaction of Aprataxin with proteins involved in the repair of both single and double strand DNA breaks (XRay Cross-Complementing 1, XRCC4 and Poly-ADP Ribose Polymerase-1) and the hypersensitivity of AOA1 patient cell lines to single and double strand break inducing agents. At the commencement of this study little was known about the in vitro and in vivo properties of Aprataxin. Initially this study focused on generation of recombinant Aprataxin proteins to facilitate examination of the in vitro properties of Aprataxin. Using recombinant Aprataxin proteins I found that Aprataxin binds to double stranded DNA. Consistent with a role for Aprataxin as a DNA repair enzyme, this binding is not sequence specific. I also report that the HIT domain of Aprataxin hydrolyses adenosine derivatives and interestingly found that this activity is competitively inhibited by DNA. This provided initial evidence that DNA binds to the HIT domain of Aprataxin. The interaction of DNA with the nucleotide hydrolase domain of Aprataxin provided initial evidence that Aprataxin may be a DNA-processing factor. Following these studies, Aprataxin was found to hydrolyse 5’adenylated DNA, which can be generated by unscheduled ligation at DNA breaks with non-standard termini. I found that cell extracts from AOA1 patients do not have DNA-adenylate hydrolase activity indicating that Aprataxin is the only DNA-adenylate hydrolase in mammalian cells. I further characterised this activity by examining the contribution of the zinc finger and FHA domains to DNA-adenylate hydrolysis by the HIT domain. I found that deletion of the zinc finger ablated the activity of the HIT domain against adenylated DNA, indicating that the zinc finger may be required for the formation of a stable enzyme-substrate complex. Deletion of the FHA domain stimulated DNA-adenylate hydrolysis, which indicated that the activity of the HIT domain may be regulated by the FHA domain. Given that the FHA domain is involved in protein-protein interactions I propose that the activity of Aprataxins HIT domain may be regulated by proteins which interact with its FHA domain. We examined this possibility by measuring the DNA-adenylate hydrolase activity of extracts from cells deficient for the Aprataxin-interacting DNA repair proteins XRCC1 and PARP-1. XRCC1 deficiency did not affect Aprataxin activity but I found that Aprataxin is destabilized in the absence of PARP-1, resulting in a deficiency of DNA-adenylate hydrolase activity in PARP-1 knockout cells. This implies a critical role for PARP-1 in the stabilization of Aprataxin. Conversely I found that PARP-1 is destabilized in the absence of Aprataxin. PARP-1 is a central player in a number of DNA repair mechanisms and this implies that not only do AOA1 cells lack Aprataxin, they may also have defects in PARP-1 dependant cellular functions. Based on this I identified a defect in a PARP-1 dependant DNA repair mechanism in AOA1 cells. Additionally, I identified elevated levels of oxidized DNA in AOA1 cells, which is indicative of a defect in Base Excision Repair (BER). I attribute this to the reduced level of the BER protein Apurinic Endonuclease 1 (APE1) I identified in Aprataxin deficient cells. This study has identified and characterised multiple DNA repair defects in AOA1 cells, indicating that Aprataxin deficiency has far-reaching cellular consequences. Consistent with the literature, I show that Aprataxin is a nuclear protein with nucleoplasmic and nucleolar distribution. Previous studies have shown that Aprataxin interacts with the nucleolar rRNA processing factor nucleolin and that AOA1 cells appear to have a mild defect in rRNA synthesis. Given the nucleolar localization of Aprataxin I examined the protein-protein interactions of Aprataxin and found that Aprataxin interacts with a number of rRNA transcription and processing factors. Based on this and the nucleolar localization of Aprataxin I proposed that Aprataxin may have an alternative role in the nucleolus. I therefore examined the transcriptional activity of Aprataxin deficient cells using nucleotide analogue incorporation. I found that AOA1 cells do not display a defect in basal levels of RNA synthesis, however they display defective transcriptional responses to DNA damage. In summary, this thesis demonstrates that Aprataxin is a DNA repair enzyme responsible for the repair of adenylated DNA termini and that it is required for stabilization of at least two other DNA repair proteins. Thus not only do AOA1 cells have no Aprataxin protein or activity, they have additional deficiencies in PolyADP Ribose Polymerase-1 and Apurinic Endonuclease 1 dependant DNA repair mechanisms. I additionally demonstrate DNA-damage inducible transcriptional defects in AOA1 cells, indicating that Aprataxin deficiency confers a broad range of cellular defects and highlighting the complexity of the cellular response to DNA damage and the multiple defects which result from Aprataxin deficiency. My detailed characterization of the cellular consequences of Aprataxin deficiency provides an important contribution to our understanding of interlinking DNA repair processes.

Neurodegenerative disorders are heterogenous in nature and include a range of ataxias with oculomotor apraxia, which are characterised by a wide variety of neurological and ophthalmological features. This family includes recessive and dominant disorders. A subfamily of autosomal recessive cerebellar ataxias are characterised by defects in the cellular response to DNA damage. These include the well characterised disorders Ataxia-Telangiectasia (A-T) and Ataxia-Telangiectasia Like Disorder (A-TLD) as well as the recently identified diseases Spinocerebellar ataxia with axonal neuropathy Type 1 (SCAN1), Ataxia with Oculomotor Apraxia Type 2 (AOA2), as well as the subject of this thesis, Ataxia with Oculomotor Apraxia Type 1 (AOA1). AOA1 is caused by mutations in the APTX gene, which is located at chromosomal locus 9p13. This gene codes for the 342 amino acid protein Aprataxin. Mutations in APTX cause destabilization of Aprataxin, thus AOA1 is a result of Aprataxin deficiency. Aprataxin has three functional domains, an N-terminal Forkhead Associated (FHA) phosphoprotein interaction domain, a central Histidine Triad (HIT) nucleotide hydrolase domain and a C-terminal C2H2 zinc finger. Aprataxins FHA domain has homology to FHA domain of the DNA repair protein 5’ polynucleotide kinase 3’ phosphatase (PNKP). PNKP interacts with a range of DNA repair proteins via its FHA domain and plays a critical role in processing damaged DNA termini. The presence of this domain with a nucleotide hydrolase domain and a DNA binding motif implicated that Aprataxin may be involved in DNA repair and that AOA1 may be caused by a DNA repair deficit. This was substantiated by the interaction of Aprataxin with proteins involved in the repair of both single and double strand DNA breaks (XRay Cross-Complementing 1, XRCC4 and Poly-ADP Ribose Polymerase-1) and the hypersensitivity of AOA1 patient cell lines to single and double strand break inducing agents. At the commencement of this study little was known about the in vitro and in vivo properties of Aprataxin. Initially this study focused on generation of recombinant Aprataxin proteins to facilitate examination of the in vitro properties of Aprataxin. Using recombinant Aprataxin proteins I found that Aprataxin binds to double stranded DNA. Consistent with a role for Aprataxin as a DNA repair enzyme, this binding is not sequence specific. I also report that the HIT domain of Aprataxin hydrolyses adenosine derivatives and interestingly found that this activity is competitively inhibited by DNA. This provided initial evidence that DNA binds to the HIT domain of Aprataxin. The interaction of DNA with the nucleotide hydrolase domain of Aprataxin provided initial evidence that Aprataxin may be a DNA-processing factor. Following these studies, Aprataxin was found to hydrolyse 5’adenylated DNA, which can be generated by unscheduled ligation at DNA breaks with non-standard termini. I found that cell extracts from AOA1 patients do not have DNA-adenylate hydrolase activity indicating that Aprataxin is the only DNA-adenylate hydrolase in mammalian cells. I further characterised this activity by examining the contribution of the zinc finger and FHA domains to DNA-adenylate hydrolysis by the HIT domain. I found that deletion of the zinc finger ablated the activity of the HIT domain against adenylated DNA, indicating that the zinc finger may be required for the formation of a stable enzyme-substrate complex. Deletion of the FHA domain stimulated DNA-adenylate hydrolysis, which indicated that the activity of the HIT domain may be regulated by the FHA domain. Given that the FHA domain is involved in protein-protein interactions I propose that the activity of Aprataxins HIT domain may be regulated by proteins which interact with its FHA domain. We examined this possibility by measuring the DNA-adenylate hydrolase activity of extracts from cells deficient for the Aprataxin-interacting DNA repair proteins XRCC1 and PARP-1. XRCC1 deficiency did not affect Aprataxin activity but I found that Aprataxin is destabilized in the absence of PARP-1, resulting in a deficiency of DNA-adenylate hydrolase activity in PARP-1 knockout cells. This implies a critical role for PARP-1 in the stabilization of Aprataxin. Conversely I found that PARP-1 is destabilized in the absence of Aprataxin. PARP-1 is a central player in a number of DNA repair mechanisms and this implies that not only do AOA1 cells lack Aprataxin, they may also have defects in PARP-1 dependant cellular functions. Based on this I identified a defect in a PARP-1 dependant DNA repair mechanism in AOA1 cells. Additionally, I identified elevated levels of oxidized DNA in AOA1 cells, which is indicative of a defect in Base Excision Repair (BER). I attribute this to the reduced level of the BER protein Apurinic Endonuclease 1 (APE1) I identified in Aprataxin deficient cells. This study has identified and characterised multiple DNA repair defects in AOA1 cells, indicating that Aprataxin deficiency has far-reaching cellular consequences. Consistent with the literature, I show that Aprataxin is a nuclear protein with nucleoplasmic and nucleolar distribution. Previous studies have shown that Aprataxin interacts with the nucleolar rRNA processing factor nucleolin and that AOA1 cells appear to have a mild defect in rRNA synthesis. Given the nucleolar localization of Aprataxin I examined the protein-protein interactions of Aprataxin and found that Aprataxin interacts with a number of rRNA transcription and processing factors. Based on this and the nucleolar localization of Aprataxin I proposed that Aprataxin may have an alternative role in the nucleolus. I therefore examined the transcriptional activity of Aprataxin deficient cells using nucleotide analogue incorporation. I found that AOA1 cells do not display a defect in basal levels of RNA synthesis, however they display defective transcriptional responses to DNA damage. In summary, this thesis demonstrates that Aprataxin is a DNA repair enzyme responsible for the repair of adenylated DNA termini and that it is required for stabilization of at least two other DNA repair proteins. Thus not only do AOA1 cells have no Aprataxin protein or activity, they have additional deficiencies in PolyADP Ribose Polymerase-1 and Apurinic Endonuclease 1 dependant DNA repair mechanisms. I additionally demonstrate DNA-damage inducible transcriptional defects in AOA1 cells, indicating that Aprataxin deficiency confers a broad range of cellular defects and highlighting the complexity of the cellular response to DNA damage and the multiple defects which result from Aprataxin deficiency. My detailed characterization of the cellular consequences of Aprataxin deficiency provides an important contribution to our understanding of interlinking DNA repair processes.

To counteract the potentially calamitous effects of genomic instability in the form of double-strand breaks (DSBs), cells have evolved with two major mechanisms. First, DNA non¬homologous end joining (NHEJ) which requires no significant homology, and second, homologous recombination (HR) that uses intact sequences on the sister chromatid or homologous chromosome as a template to repair the broken DNA. Although NHEJ repairs DSBs in all stages of cell cycle; it is generally error-prone due to insertions or deletions of few nucleotides at the breakpoint. In contrast, DSBs that are generated during S and G2 phase of the cell are preferentially repaired by HR that utilizes neighboring sister chromatid as a template. A central role in the HR reaction is promoted by the RAD51 recombinase which polymerizes onto single-stranded DNA (ssDNA), catalyzes pairing and strand invasion with homologous DNA molecule. Assembly of RAD51 monomers onto ssDNA is a relatively slow process and is facilitated by several mediator proteins. The tumor suppressor protein BRCA2 is the best-characterized RAD51 mediator in DSB repair by HR. Many reports in the past two decades have established that RAD51 recruitment at break sites also depends on the RAD51 paralogs. Mammalian cells encode five RAD51 paralogs; RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 which share 20–30% identity at amino acid level with RAD51 and with each other. In addition to their role in HR, RAD51 paralogs have been identified to be involved in DNA damage signaling and replication fork maintenance. In addition, mouse knockout of RAD51 paralogs causes early embryonic lethality. Recent studies show that germline mutations in all five RAD51 paralogs cause various types of cancer including breast and ovarian cancers. Pedigree analyses revealed that similar to BRCA1 and BRCA2, pathological missense mutants of RAD51C were of high penetrance. Historically, defects in the DNA repair pathways have been exploited for cancer chemo-and radiotherapy. In an attempt to develop better cancer therapeutic approaches, the concept of synthetic lethality for cancer therapy has been recently proposed. One such example is the use of PARP1 inhibitors to treat tumors carrying mutations in HR genes, such as BRCA1 and BRCA2. Inhibition of PARP1 compromises single-strand break repair (SSBR) pathway. Upon replication fork collision, the accumulated SSBs are converted to one-ended DSBs, which are efficiently repaired by the HR for cell survival. As a result, HR-deficient tumors with BRCA1-or BRCA2-deficiency exhibit extreme sensitivity to PARP-1 inhibition resulting in cell death. This approach was highly successful in targeting tumors with severe defects in Fanconi anemia (FA)-BRCA proteins which led to PARP inhibitors being tested in clinical trials. However, targeting cancer cells that express hypomorphic mutants of HR proteins is highly challenging since such partially functional mutants require a high dosage of PARP inhibitors for effective sensitization which renders normal cells toxic and can also lead to tumor resistance. The pathological RAD51C mutants that were identified in breast and ovarian cancer patients are hypomorphic with partial repair function. The first part of my Ph.D. thesis is aimed at developing an effective strategy to target cells that express hypomorphic RAD51C mutants. To this end, we used RAD51C deficient CL-V4B hamster cells and expressed the pathological RAD51C mutants associated with breast and ovarian cancers. Cells expressing RAD51C mutants that were severely defective for HR function exhibited high sensitivity to low doses of PARP1 inhibitor (4-ANI). These cells also accumulated in G2/M and displayed chromosomal aberrations. However, RAD51C mutants that were hypomorphic were partially sensitized even at higher concentrations of PARP inhibitor. RAD51C/ CL-V4B cells displayed higher PARP activity compared WT V79B cells. Notably, PARP activity was directly proportional to the sensitivity of RAD51C mutants towards 4-ANI where highly sensitive RAD51C mutants showed higher PARP activity and vice versa. Increased PARP activity was associated with replication stress as confirmed by an increase of PARP activity in cells treated with replication stress inducer, hydroxyurea (HU). Notably, treatment of CL-V4B cells with PARP1 inhibitor (4-ANI) resulted in the accumulation of PARP1 onto the chromatin which eventually led to the formation of DSBs which suggests that PARP1 entrapment triggers replication fork collapse leading to one-ended DSBs in S-phase. To further understand the molecular mechanism of PARP inhibitor-induced toxicity of RAD51C deficient cells, we carried out chromatin fractionation from V79B and CL-V4B cells at varying time points of 4-ANI treatment. Surprisingly, there was an enhanced loading of NHEJ proteins on chromatin in CL-V4B compared to V79B cells. Consistently, an increased error-prone NHEJ was observed in CL-V4B cells which resulted in increased chromosomal aberrations and cell death. Furthermore, inhibition of DNA-PKcs or depletion of KU70 or Ligase IV restored this phenotype. Thus, error-prone NHEJ in collaboration with PARP inhibition sensitizes RAD51C deficient cells. Since ionizing radiation (IR) is known to stimulate NHEJ activity, we hypothesized that irradiation in combination with PARP inhibitor would further sensitize the RAD51C deficient tumors. Strikingly, stimulation of NHEJ by a low dose of IR in the PARP inhibitor-treated RAD51C deficient cells and cells expressing pathological RAD51C mutants induced enhanced toxicity ‘synergistically’. These results demonstrate that cancer cells arising due to hypomorphic mutations in RAD51C can be specifically targeted by a ‘synergistic approach’ and imply that this strategy can be potentially applied to cancers with hypomorphic mutations in other HR pathway genes. In addition to nuclear functions, RAD51 paralogs RAD51C and XRCC3 have been shown to localize to mitochondria and contribute to mitochondrial genome stability. However, the molecular mechanism by which RAD51 and RAD51 paralogs carry out this function is unclear. The second part of my thesis was dedicated to studying whether RAD51C/XRCC3 facilitates mitochondrial DNA replication and the underlying mechanism by which RAD51C/XRCC3 participate in mitochondrial genome maintenance during unperturbed conditions. Using mitochondrial subfractionation we show that RAD51 and RAD51 paralogs (RAD51C and XRCC3) are an integral part of mitochondrial nucleoid and absence of RAD51C/XRCC3 and RAD51 prevents the restoration of mtDNA upon depletion of mtDNA. This suggests that RAD51 and RAD5C/XRCC3 participate in mtDNA replication. To determine whether this function of RAD51C is exclusive to mitochondria we expressed NLS mutant of RAD51C which was defective for nuclear functions. Interestingly, cells expressing RAD51C R366Q were able to efficiently repopulate the depleted mtDNA after EtBr stress similar to that of WT RAD51C expressing cells, suggesting a nuclear independent function of RAD51C in mitochondrial genome maintenance. mtDNA-IP analysis revealed that RAD51 and RAD51C/XRCC3 are recruited to the mtDNA control regions spontaneously along with mitochondrial polymerase POLG. Moreover, RAD51 was found to associate with TWINKLE helicase and this association was required for the recruitment of RAD51 and RAD51C/XRCC3 at the D-loop. As in nucleus, mtDNA replisome also encounters replication stresses like altered dNTP pools, a collision between replication and transcription machinery, rNTP incorporation, oxidative stress which hampers replication fork progression. Using Dideoxycytidine (ddC) as replication stress inducer in mitochondria, we observed nearly 3-4 fold enrichment of RAD51, RAD51C, XRCC3 and POLG at the mtDNA mutation hotspot region D310. Notably, RAD51C/XRCC3 deficient cells exhibited increased lesions in the mitochondrial genome spontaneously, pointing towards the importance of RAD51C/XRCC3 in the prevention of mtDNA lesions. Moreover, RAD51C/XRCC3 deficiency prevented the repair of ddC induced mtDNA lesions. Given that RAD51C/XRCC3 and RAD51 are localized to mtDNA control regions along with POLG and their deficiency affects mtDNA replication we were curious to learn the effect of RAD51C/XRCC3 deficiency on the recruitment of POLG in mtDNA. To test this we performed a mtDNA-IP assay of POLG in RAD51C deficient cells which revealed that deficiency of RAD51C/XRCC3 and RAD51 affected the recruitment of POLG on mtDNA control regions. As a consequence RAD51C/XRCC3 deficient cells exhibit aberrant mitochondrial functions. These findings propose a mechanism for a direct role of RAD51C/XRCC3 in maintaining the mtDNA integrity under replication stress conditions.

Genomic integrity is a fundamental requisite for survival and proliferation of all organisms. The genetic material is continuously threatened by a multitude of extrinsic and intrinsic factors. Consequently, the presence of strong DNA repair systems is essential to aid errorfree transmission of genetic material to successive generations. In prokaryotes, repair by homologous recombination (HR) provides a major means to reinstate the genetic information lost in DNA damage. Pathogenic bacteria, such as Mycobacterium tuberculosis, face an additional threat of DNA damage due to antibiotic treatment and immune stresses inside the macrophage. Consequently, M. tuberculosis has evolved a remarkably strong DNA repair network, providing it robust survivability in the harsh environments faced inside the host cell. The importance of HR or recombination repair in pathogenesis and emergence of antibiotic resistance in M. tuberculosis is well established. However, many aspects of the pathway remain elusive as of now. This thesis is concerned with the analysis of recombination repair system in the genus mycobacteria and characterization of two novel proteins identified in the process. Chapter 1 gives a detailed account of the mechanics of HR, using the well studied E. coli system and highlights the differences in mycobacteria. Recombination repair comprises a series of processes carried out by more than 20 proteins, that ultimately leads to repair of damaged DNA. Processing of DNA strands at double strand breaks and single strand gaps, to produce a 3' overhang, initiates the process. Unlike E. coli, complexes of RecFO-RecR and AdnAB-RecR provide two alternate pathways for end resection of strands and RecA loading in mycobacteria. The exchange of an undamaged strand with the damaged strand, facilitated by RecA, is central to recombination. Additionally, Single-Stranded DNA binding proteins (SSB) facilitate the loading of RecA onto the single-stranded overhang produced by the pre-processing enzymes. Resolution of strands formed due to strand exchange, via multi-stand branched DNA intermediates (such as D-loops, three-way junctions, Holliday junctions etc) by RuvABC or RecG, is the final step of recombination. An additional HJ resolvase YqgF, with unclear functions, is also present in mycobacteria. Furthermore, the major end resection enzymes (RecBCD) involved in HR in E. coli, were implicated in the Single-Strand Annealing (SSA) pathway in mycobacteria. As part of an effort to improve the understanding of recombination repair in mycobacteria, a structure based genomic search for such proteins was carried out in 43 mycobacteria with known genome sequences (Chapter 2). Of about 20 proteins known to be involved in the pathway, a set of 9 proteins, namely, RecF, RecO, RecR, RecA, SSBa, RuvA, RuvB and RuvC was found to be indispensable among the 43 mycobacterial strains. A domain level analysis indicated that most domains involved in recombination repair are unique to these proteins and are present as single copies in the genomes. Synteny analysis reveals that the gene order of proteins involved in the pathway is not conserved, suggesting that they may be regulated differently in different species. Sequence conservation among the same protein from different strains suggests the importance of RecO-RecA and RecFORRecA presynaptic pathways in the repair of double strand-breaks and single strand-breaks respectively. New insights into the binding of small molecules to the relevant proteins are provided by binding pocket analysis using three-dimensional structural models. New annotations obtained from the analysis, include identification of a protein (RecGwed) with a probable Holliday junction binding role present in 41 mycobacterial genomes and that of a RecB-like nuclease, containing a cas4 domain, present in 42 genomes. A second SingleStranded DNA Binding protein (SSBb), in addition to the canonical one (SSBa), was present in all mycobacteria except M. leprae. Chapter 3 describes the cloning, expression, purification and structural studies on SSBb from M. smegmatis (MsSSBb). MsSSBb has been crystallized and X-ray analyzed in the first structure elucidation of a mycobacterial SSBb. The protein crystallizes in hexagonal space group P6522 (a = b =73.61 Å, c = 216.21 Å), with half a tetrameric molecule in the asymmetric unit of the cell. In spite of the low sequence identity between SSBas and SSBbs in mycobacteria, the tertiary and quaternary structure of the DNA binding domain of MsSSBb is similar to that observed in mycobacterial SSBas. In particular, the quaternary structure is 'clamped' using a C-terminal stretch of the N-domain, which endows the tetrameric molecule with additional stability and its characteristic shape. A comparison involving available, rather limited, structural data on SSBbs from other sources, appears to suggest that SSBbs could exhibit higher structural variability than SSBas do. It was realized that many bacterial species have a paralogous SSBb. The SSBb proteins have not been well characterized. While in B. subtilis, SSBb has been shown to be involved in genetic recombination; in S. coelicolor it mediates chromosomal segregation during sporulation. Chapter 4 describes the distinct properties and the role of SSBb in mycobacteria. Sequence analysis of SSBs from mycobacterial species suggests low conservation of SSBb proteins, as compared to the conservation of SSBa proteins. Like most bacterial SSB proteins, M. smegmatis SSBb (MsSSBb) forms a stable tetramer. However, solution studies indicate that MsSSBb is less stable towards thermal and chemical denaturation than MsSSBa. Also, in contrast to the 5-20 fold differences in DNA binding affinity between paralogous SSBs observed in other organisms, MsSSBb is only about twofold poorer in its DNA binding affinity than MsSSBa. The expression levels of ssbB gene increased during UV and hypoxic stresses, while the levels of ssbA expression declined. A direct physical interaction of MsSSBb and RecA, mediated by the C-terminal tail of MsSSBb was also established. The results obtained in this study indicate a role of MsSSBb in recombination repair during stress. Chapter 5 describes the characterization of the previously annotated hypothetical protein RecGwed and its probable role as a novel regulator in the resolution of branched DNA structures. The protein is composed of an unusually charged N-terminus and a C-terminal 'wedge' domain, similar to the wedge domain of RecG. A database search suggested that RecGwed is predominately present in the phylum Actinobacteria, along with some other known human pathogens. Purified M. smegmatis RecGwed (MsRecGwed) exists as a stable monomer in the solution. CD studies and homology modeling indicated an unusually low content of regular secondary structures. MsRecGwed was able to bind branched DNA structures such as Holliday junction, three-way junction, three-strand junction and replication fork in vitro, while it does not interact with ss- or dsDNA. The expression of recGwed in M. smegmatis was up-regulated during stationary phase/UV damage and down-regulated during MMS/H2O2 treatment. These observations indicate the possibility of involvement of RecGwed in DNA transactions in post-replicative (stationary phase) recombination events, that proceed though branched DNA intermediates. The work described in this chapter is the first report of characterization of RecGwed-like proteins. Taken together, the work done in this thesis augments the existing repertoire of proteins known to be involved in DNA repair pathways in mycobacteria. As indicated in the concluding chapter, this study also creates a trail of future experiments that will improve our current understanding of HR in mycobacteria. As a part of ongoing efforts in the laboratory, on the characterization of enzymes which sanitize the nucleotide pool to prevent DNA damage, structural studies on M. smegmatis MutT2 have been carried out (Appendix). Structure of the native protein, and its complexes with substrates 5me-dCTP, dCTP and CTP and the respective products, has been determined. The work presented here is the first report of MutT2-type CTP pyrophosphorylase enzymes in complex with substrates. It provides insights into the mechanism of action and the molecular basis of the functioning of mycobacterial MutT2

DNA damage produced in C3H/HeN murine tissues by ionizing radiation was characterized at the level of the individual cell with the goal of defining tissue dependent differences in DNA single-strand break (SSB) induction and repair. Subsequently, nicotinamide, was examined as a modifier of initial DNA damage and SSB rejoining following irradiation. The alkaline comet assay, a single-cell gel electrophoresis method, was used to examine cells from SCCVM tumours, spleen, bone marrow, liver, jejunum, testis, thymus and cerebellum. Cells from all tissues irradiated in vitro showed similar radiosensitivity. However, for in vivo irradiation, rapid SSB rejoining which occurs in cells during irradiation led to differences between tissues. Also, tumour and testis showed less damage in vivo than other normal tissues. Consistent with previous studies, these two tissues were found to contain radiobiologically hypoxic cells. Efficiency of SSB rejoining was cell type-dependent; cells from SCCVII tumors rejoined breaks about 5 times more rapidly than cells from cerebellum. Heterogeneity in speed of rejoining was minimal among cells of a tissue, and no significant damage remained 4 hours following 15 Gy. However, extensive DNA degradation was observed in all tissues except brain 48 hours after 15 Gy. DNA ladder patterns in agarose gels, typical of apoptosis, were observed 4 hours after 2-10 Gy in spleen and thymus. The vitamin B analogue, nicotinamide, was shown to improve testis and tumour oxygenation, in agreement with other studies. A new observation was that nicotinamide (500 mg/kg or more) given before irradiation inhibited SSB rejoining in cells of all tissues except brain. Furthermore, radiation-induced DNA degradation was found to be greatly accelerated by nicotinamide. Both effects are likely to involve poly(ADP-ribose) polymerase inhibition. However, while nicotinamide significantly retarded radiation-induced SSB rejoining in tumors, the biological significance of this effect is questionable since nicotinamide did not enhance oxygen-independent killing of irradiated tumor cells.