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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Γενετική ανάλυση στον κληρονομικό καρκίνο του μαστού

Τσιτλαΐδου, Μαριάνθη 07 July 2015 (has links)
O καρκίνος του μαστού αποτελεί την δεύτερη πιο συχνή μορφή καρκίνου που απαντάται και στα δύο φύλα ενώ είναι συνιστά την πιο συχνά εμφανιζόμενη μορφή καρκίνου στις γυναίκες καθώς και την πρώτη αιτία θανάτου στο γυναικείο φύλο. Η γενετική ανάλυση του κληρονομικού καρκίνου έχει ενταχθεί τα τελευταία χρόνια και αποτελεί σημαντικό μέρος της κλινικής πράξης, καθώς όλο και περισσότεροι ασθενείς ελέγχονται για την ύπαρξη γαμετικών μεταλλάξεων στα υψηλής διεισδυτικότητας γονίδια BRCA1 και BRCA2. Προκειμένου να μελετήσουμε την συνεισφορά των γονιδίων BRCA1 και BRCA2 στην εμφάνιση καρκίνου μαστού στον ελληνικό πληθυσμό εξετάστηκαν 200 άτομα που παρουσίαζαν σημαντικό ατομικό ή/και οικογενειακό ιστορικό καρκίνου του μαστού ή/και των ωοθηκών. Έτσι, στην περίπτωση του γονίδιου BRCA1 βρέθηκαν 13 διαφορετικές παθογόνοι μεταλλάξεις σε 29 από τις ασθενείς (14.5%) ενώ για το γονίδιο BRCA2 αναλύθηκαν συνολικά 156 ασθενείς (155 γυναίκες και 1 άνδρας) και μεταλλάξεις ανιχνεύθηκαν σε 8 γυναίκες (5.2%) και 1 άνδρα (100%). Παράλληλα έγινε προσπάθεια χαρακτηρισμού του φάσματος μεταλλάξεων του μετρίου διεισδυτικότητας γονιδίου RAD51C σε ελληνικές οικογένειες. Η ανάλυση αυτή πραγματοποιήθηκε σε 87 ασθενείς όμως δεν ανιχνεύθηκε κάποια παθογόνος μετάλλαξη σε κάποιον ασθενή. Επιπρόσθετα, πραγματοποιήθηκε η ανίχνευση μεγάλων γονιδιακών αναδιατάξεων στα γονίδια BRCA1 και BRCA2 με την βοήθεια τριών τεχνικών ανίχνευσης: την τεχνική της ανίχνευσης γονιδιακών αναδιατάξεων με χρήση διαγνωστικών εκκινητών για τις πιο συχνές γονιδιακές αναδιατάξεις του γονιδίου BRCA1 στον ελληνικό πληθυσμό (δύο απαλειφές του εξωνίου 20, μία απαλειφή των εξονίων 23 και 24 και τέλος μια απαλειφή του εξονίου 24), την τεχνική της πολλαπλής και ποσοτικής αλυσιδωτής αντίδρασης της πολυμεράσης μικρών φθοριζόντων τμημάτων (QMPSF) για το γονίδιο BRCA1 και την τεχνική της πολλαπλής ενίσχυσης τμημάτων DNA μέσω ιχνηθετών (MLPA®) τόσο για το γονίδιο BRCA1 όσο και για το γονίδιο BRCA2. Η τεχνική της ανίχνευσης με την χρήση διαγνωστικών εκκινητών εφαρμόστηκε σε 200 ασθενείς με καρκίνο μαστού ή/και ωοθηκών και ανιχνεύθηκαν 15 ασθενείς που έφεραν κάποια από τις συχνότερες γονιδιακές αναδιατάξεις του γονιδίου BRCA1 (7.5%). Αντίθετα, με την τεχνική του QMPSF και του MLPA δεν ανιχνεύθηκε κάποια γονιδιακή αναδιάταξη στα BRCA1/2 στους συνολικά 24 ασθενείς που εξετάστηκαν. Επιπλέον, στα πλαίσια της παρούσας διατριβής πραγματοποιήθηκε η ανάλυση 403 γυναικών με τριπλά αρνητικό καρκίνο του μαστού ανεξαρτήτως οικογενειακού ιστορικού και ηλικίας εμφάνισης της νόσου. Κατά την ανάλυση αυτή ελέγχθηκαν συγκεκριμένα εξόνια καθώς και γονιδιακές αναδιατάξεις του BRCA1. Μεταλλάξεις ανιχνεύθηκαν σε 65 από τις ασθενείς αυτές (16%). Στα πλαίσια της παρούσας διατριβής πραγματοποιήθηκε ανάλυση απλοτύπου προκειμένου να αποδειχθεί ότι η απαλειφή των εξονίων 23 και 24 αποτελεί ελληνική ιδρυτική μετάλλαξη. Για το σκοπό αυτό αναλύθηκε ο απλότυπος σε 21 οικογένειες που έφεραν την συγκεκριμένη γονιδιακή αναδιάταξη με την βοήθεια της τεχνικής των πολυμορφικών δεικτών επαναλήψεων μικροδορυφορικού DNA. Η ανάλυση απλοτύπου κατέδειξε ότι 8 από 10 γενετικούς τόπους είναι κοινοί μεταξύ των ατόμων που φέρουν την απαλοιφή επιβεβαιώνοντας έτσι την υπόθεση ότι η απαλειφή αυτή αποτελεί ιδρυτική μετάλλαξη του ελληνικού πληθυσμού. Ακολούθως, μελετήθηκε η συνεισφορά και άλλων γονιδίων στην προδιάθεση για καρκίνο μαστού και ωοθηκών και πραγματοποιήθηκε ανάλυση 21 γονιδίων (BRCA1, BRCA2, CHEK2, PALB2, BRIP1, TP53, PTEN, STK11, CDH1, ATM, BARD1, MLH1, MRE11, MSH2, MSH6, MUTYH, NBN, PMS1, PMS2, RAD50, RAD51C) με την χρήση τεχνολογίας αλληλούχισης επόμενης γενεάς. Για το σκοπό αυτό αναλύθηκαν 42 ασθενείς με σοβαρό ιστορικό καρκίνο μαστού ή/και ωοθηκών, οι οποίες είχαν βρεθεί αρνητικές για μεταλλάξεις στα γονίδια BRCA1 & 2. Κατά την ανάλυση αυτή βρέθηκαν μεταλλάξεις σε 11 ασθενείς (26.2%). Ακόμα, πραγματοποιήθηκε εξομική αλληλούχιση σε μια ασθενή, η οποία παρουσίαζε βαρύτατο ατομικό και οικογενειακό ιστορικό και είχε βρεθεί αρνητική για μεταλλάξεις σε 21 γονίδια (BRCA1, BRCA2, CHEK2, PALB2, BRIP1, TP53, PTEN, STK11, CDH1, ATM, BARD1, MLH1, MRE11, MSH2, MSH6, MUTYH, NBN, PMS1, PMS2, RAD50, RAD51C). Η αλληλούχιση αυτή πραγματοποιήθηκε από την εταιρεία BGI. Συνολικά ανιχνεύθηκαν 39.762 πολυμορφισμοί ενός νουκλεοτιδίου (SNPs) και 1,646 μικρές ενθέσεις/απαλειφές (InDels) σε ολόκληρο το γονιδίωμα της ασθενούς. Μετά από ανάλυση των αποτελεσμάτων αυτών καταλήξαμε σε 12 παραλλαγές που αποτελούν ενδεχομένως πιθανά παθογόνα αλληλόμορφα. / Breast cancer is the second most common cancer in both sexes while it is the most frequent cancer as well as the first cause of death in women. The genetic testing of hereditary cancer is part of the everyday clinic as the number of patients that is checked for germline mutation in BRCA1 and BRCA2 is continuously increasing. In order to study the contribution of the high penetrance genes BRCA1 and BRCA2 in breast cancer in Greece 200 patients with severe history of breast and/or ovarian cancer were screened. In BRCA1 13 different pathogenic mutations were found in 29 patients (14.5%) while in BRCA2 156 patients were screened (155 female and 1 male) and pathogenic mutations were identified in 8 female (5.2%) and 1 male patients (100%). At the same time, we tried to characterize the mutation spectrum of the medium penetrance gene RAD51C in Greek families. For this purpose, we screened 87 patients but no mutations was found. In addition, the identification of large genomic rearrangements in both genes took place, using three methods: diagnostic primers were used in order to identify the 4 most common BRCA1 genomic rearrangements in Greece (two deletions in exon 20, one deletion of exons 23 and 24 and one in exon 24), the QMPSF method for the BRCA1 genomic rearrangements (Quantitative Multiplex PCR of Short Fluorescent fragments) and the MLPA method for both BRCA1 and BRCA2 genomic rearrangements (Multiplex Ligation-dependent Probe Amplification). With the first technique 200 patients with breast and/or ovarian cancer were analyzed and 15 of them were found to carry one of the most frequent BRCA1 genomic rearrangements. In the contrary, no large genomic rearrangements in neither gene was identified among the 24 patients who were screened using the two latter methods (QMPSF and MLPA). Also, we have screened 403 triple negative female patients who were diagnosed with triple-negative invasive breast cancer, independently of their age or family history, for germline BRCA1 mutations in exons where a mutation have previously been found in Greek population including the genomic rearrangements. Mutation were identified in 65 patients (16%). In this study we used haplotype analysis in order to demonstrate that the BRCA1 deletion of exons 23 and 24 constitutes a Greek founder mutation. For this purpose, we performed haplotype analysis in 21 Greek families who carry this specific genomic rearrangement using the DNA Microsatellite Repeats analysis. The haplotype analysis showed that 8 out of 10 genetic regions are common among people that carry this deletion confirming that the deletion is a Greek founder mutation. We also studied the contribution of other genes in breast/ovarian cancer predisposition by studying 21 genes (BRCA1, BRCA2, CHEK2, PALB2, BRIP1, TP53, PTEN, STK11, CDH1, ATM, BARD1, MLH1, MRE11, MSH2, MSH6, MUTYH, NBN, PMS1, PMS2, RAD50, RAD51C) using next generation sequencing techniques. We examined 42 patients with severe breast and/or ovarian cancer history that were found negative for BRCA1/2 mutations. In this analysis pathogenic mutation were found in 11 patients (26.2%). At last, exome sequencing was performed in a female patient who had severe family history and was found negative for germline mutations in 21 genes (BRCA1, BRCA2, CHEK2, PALB2, BRIP1, TP53, PTEN, STK11, CDH1, ATM, BARD1, MLH1, MRE11, MSH2, MSH6, MUTYH, NBN, PMS1, PMS2, RAD50, RAD51C). The sequencing was carried out by Beijing Genomics Institute (BGI). In total, were identified 39.762 singe nucleotide polymorphisms (SNPs) and 1.646 insertions - deletions (InDels) in the patient's exome. After the result analysis we concluded in 12 variants that could constitute possible pathogenic alleles.
2

The Rad51 family of proteins: Interactions, vitamin D, and implications in head and neck cancer

Lu, Daniel Kee 01 January 2013 (has links)
Protection of the genome from carcinogenic consequences of DNA double-strand breaks (DSBs) is accomplished through the pathways of non-homologous end-joining (NHEJ) or homologous recombinational repair. Five human proteins with homology to Rad51 known as the Rad51 paralogs, Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3, whose loss of function in cell lines leads to high chromosomal instability. Previous studies have shown Rad51C participate in two paralog protein complexes, one containing Rad51B, Rad51C, Rad51D and XRCC2 (BCDX2) and the other containing only Rad51C and XRCC3 (CX3). However, the only structural data available is the crystal structure of RecA, the bacterial homolog, the determination of the N-terminus of human Rad51 by NMR, and the crystal structure of Pyroccocus furious Rad51. Currently the Alvinlla pompejana Rad51C has been cloned, expressed and is currently being crystallized in the Tainer laboratory (UC Berkeley) since the human Rad51C protein has proven too difficult to be utilized. To test functional association of Hs Rad51B and Hs XRCC3 to Ap Rad51C. The human proteins were heterologously expressed in Pichia pastoris and the other expressed in E. coli. The proteins were extracted and interaction was tested through co-immunoprecipitation. Initial results depict weak binding or an unstable interaction between Hs Rad51B and Ap Rad51C. Hs XRCC3 and Ap Rad51C interaction remains unclear and requires further testing. Additionally, we have utilized a cellular model of HNSCC to identify whether the down-regulation of Rad51 after application of VD 3 is concomitant with the down-regulation of NBS1. NBS1 is a DNA repair protein involved in both pathways of DNA double-strand break repair, non-homologous end-joining and homologous recombinational repair. It has recently been demonstrated that NBS1 binds to Rad51 aiding in its localization to sites of DNA damage. VD 3 is a potential chemopreventive agent in the treatment of head and neck cancer. For the in vitro model Rad51 and NBS1 protein were both extracted from SCC25 and MCF-7 cancer cell lines were treated with 100 nM of VD 3 . For the in vivo model hamsters cheek pouch tissue sections with VD 3 treated and DMBA over the course of 14 weeks were used. Rad51 and NBS1 staining is restricted to the nuclei of the basal cell layer of the epithelium in VD 3 treated animals as compared to untreated controls where staining is evident throughout the dysplastic epithelium and is not restricted to nuclei. Unlike the western blot data of Rad51 that shows similar downregulation as the immunocytochemistry, the western blot analysis of NBS1 is unclear. However, the immunocytochemistry suggests that NBS1 is also downregulated by VD 3 in vivo, and therefore, it may be implied that both the HRR and NHEJ pathways are involved in the cellular effects of VD 3 in HNSCC.
3

Etude des acteurs et des interactions entre les voies de recombinaison chez Arabidopsis thaliana / Study of the actors and of the interactions between the recombination pathways of Arabidopsis thaliana

Serra, Heïdi 05 September 2014 (has links)
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.
4

Role of Mammalian RAD51 Paralogs in Genome Maintenance and Tumor Suppression

Somyajit, Kumar January 2014 (has links) (PDF)
My research was focused on understanding the importance of mammalian RAD51 paralogs in genome maintenance and suppression of tumorigenesis. The investigation carried out during this study has been addressed toward gaining more insights into the involvement of RAD51 paralogs in DNA damage signalling, repair of various types of lesions including double stranded breaks (DSBs), daughter strand gaps (DSGs), interstrand crosslinks (ICLs), and in the protection of stalled replication forks. My study highlights the molecular functions of RAD51 paralogs in Fanconi anemia (FA) pathway of ICL repair, in the ATM and ATR mediated DNA damage responses, in homologous recombination (HR), and in the recovery from replication associated lesions. My research also focused on the development of a novel photoinducible ICL agent for targeted cancer therapy. The thesis has been divided into following sections as follows: Chapter I: General introduction that describes about DNA damage responses and the known functions of RAD51 paralogs across species in DNA repair and checkpoint The genome of every living organism is susceptible to various types of DNA damage and mammalian cells are evolved with various DNA damage surveillance mechanisms in response to DNA damages. In response to DNA damage, activated checkpoints arrest the cell cycle progression transiently and allow the repair of damaged DNA. Upon completion of DNA repair, checkpoints are deactivated to resume the normal cell cycle progression. Defective DNA damage responses may lead to chromosome instability and tumorigenesis. Indeed, genome instability is associated with several genetic disorders, premature ageing and various types of cancer in humans. The major cause of chromosome instability is the formation of DSBs and DSGs. Both DSBs and DSGs are the most dangerous type of DNA lesions that arise endogenously as well as through exogenous sources such as radiations and chemicals. Spontaneous DNA damage is due to generation of reactive oxygen species (ROS) through normal cellular metabolism. Replication across ROS induced modified bases and single strand breaks (SSBs) leads to DSGs and DSBs, respectively. Such DNA lesions need to be accurately repaired to maintain the integrity of the genome. To understand the various cellular responses that are triggered after different types of DNA damage and the possible roles of RAD51 paralogs in these processes, chapter I of the thesis has been distributed in to multiple sections as follows: Briefly, the initial portion of the chapter provides a glimpse of various types of DNA damage responses and repair pathways to deal with the lesions arising from both endogenous as well as exogenous sources. Owing to the vast range of cellular responses and pathways, the following section provides the detailed description and mechanisms of various pathways involved in taking care of wide range of DNA lesions from SSBs to DSBs. Subsequent section of chapter I provides a comprehensive description of maintenance of genome stability at the replication fork and telomeres. Germline mutations in the genes that regulate genome integrity cause various genetic disorders and cancer. Mutations in ATM, ATR, MRE11, NBS1, BLM and FANC (1-16), BRCA1 and BRCA2 that are known to regulate DNA damage signaling, DNA repair and genome integrity lead to chromosome instability disorders such as ataxia-telangiectasia, ATR-Seckel syndrome, AT-like disorder, Nijmegen breakage syndrome, Bloom syndrome, FA, and breast and ovarian cancers respectively. Interestingly, RAD51 paralog mutations are reported in patients with FA-like disorder and various types of cancers including breast and ovarian cancers. Mono-allelic germline mutations in all RAD51 paralogs are reported to cause cancer in addition to the reported cases of FA-like disorder with bi-allelic germline mutations in RAD51C and XRCC2. In accordance, the last section of the chapter has been dedicated to describe the genetics of breast and ovarian cancers and the known functions of tumor suppressors such as BRCA1, BRCA2 and RAD51 paralogs in the protection of genome. Despite the identification of five RAD51 paralogs nearly two decades ago, the molecular mechanism(s) by which RAD51 paralogs regulate HR and genome maintenance remain obscure. To gain insights into the molecular mechanisms of RAD51 paralogs in DNA damage responses and their link with genetic diseases and cancer, the following objectives were laid for my PhD thesis: 1) To understand the functional role of RAD51 paralog RAD51C in FA pathway of ICL repair and DNA damage signalling. 2) To dissect the ATM/ATR mediated targeting of RAD51 paralog XRCC3 in the repair of DSBs and intra S-phase checkpoint. 3) To uncover the replication restart pathway after transient replication pause and the involvement of distinct complexes of RAD51 paralogs in the protection of replication forks. 4) To design photoinducible ICL agent that can be activated by visible light for targeted cancer therapy. Chapter II: Distinct roles of FANCO/RAD51C protein in DNA damage signaling and repair: Implications for Fanconi anemia and breast cancer susceptibility RAD51C, a RAD51 paralog has been implicated in HR. However, the underlying mechanism by which RAD51C regulates HR mediated DNA repair is elusive. In 2010, a study identified biallelic mutation in RAD51C leading to FA-like disorder, whereas a second study reported monoallelic mutations in RAD51C associated with increased risk of breast and ovarian cancers. However, the role of RAD51C in the FA pathway of DNA cross-link repair and as a tumor suppressor remained obscure. To understand the role of RAD51C in FA pathway of ICL repair and DNA damage response, we employed genetic, biochemical and cell biological approaches to dissect out the functions of RAD51C in genome maintenance. In our study, we observed that RAD51C deficiency leads to ICL sensitivity, chromatid-type errors, and G2/M accumulation, which are hallmarks of the FA phenotype. We found that RAD51C is dispensable for ICL unhooking and FANCD2 monoubiquitination but is essential for HR, confirming the downstream role of RAD51C in ICL repair. Furthermore, we demonstrated that RAD51C plays a vital role in the HR-mediated repair of DSBs associated with replication. Finally, we showed that RAD51C participates in ICL and DSB induced DNA damage signaling and controls intra-S-phase checkpoint through CHK2 activation. Our analyses with pathological mutants of RAD51C displayed that RAD51C regulates HR and DNA damage signaling distinctly. Together, these results unravel the critical role of RAD51C in the FA pathway of ICL repair and as a tumor suppressor. Chapter III: ATM-and ATR-mediated phosphorylation of XRCC3 regulates DNA double-strand break-induced checkpoint activation and repair The RAD51 paralogs XRCC3 and RAD51C have been implicated in HR and DNA damage responses, but the molecular mechanism of their participation in these pathways remained obscured. In our study, we showed that an SQ motif serine 225 in XRCC3 is phosphorylated by ATR kinase in an ATM signaling pathway. We found that RAD51C in CX3 complex but not in BCDX2 complex is essential for XRCC3 phosphorylation, and this modification follows end resection and is specific to S and G2 phases. XRCC3 phosphorylation was found to be required for chromatin loading and stabilization of RAD51 and HR-mediated repair of DSBs. Notably, in response to DSBs, XRCC3 participates in the intra-S-phase checkpoint following its phosphorylation and in the G2/M checkpoint independently of its phosphorylation. Strikingly, we found that XRCC3 distinctly regulates recovery of stalled and collapsed replication forks such that phosphorylation was required for the HR-mediated recovery of collapsed replication forks but is dispensable for the recovery of stalled replication forks. Together, our findings suggest that XRCC3 is a new player in the ATM/ATR-induced DNA damage responses to control checkpoint and HR-mediated repair. Chapter IV: RAD51 paralogs protect stalled forks and mediate replication restart in an FA-BRCA independent manner Mammalian RAD51 paralogs RAD51 B, C, D, XRCC2 and XRCC3 are critical for genome maintenance. To understand the crucial roles of RAD51 paralogs during spontaneously arising DNA damage, we have studied the RAD51 paralogs assembly during replication and examined the replication fork stability and its restart. We found that RAD51 paralogs are enriched onto the S-phase chromatin spontaneously. Interestingly, the number of 53BP1 nuclear bodies in G1-phase and micro-nucleation which serve as markers for under replicated lesions increases after genetic ablation of RAD51C, XRCC2 and XRCC3. Furthermore, we showed that RAD51 paralogs are specifically enriched at two major fragile sites FRA3B and FRA16D after replication fork stalling. We found that all five RAD51 paralogs bind to nascent DNA strands after replication fork stalling and protect the fork. Nascent replication tracts created before fork stalling with hydroxyurea degrade in the absence of RAD51 paralogs but remain stable in wild-type cells. This function was dependent on ATP binding at the walker A motif of RAD51 paralogs. Our results also suggested that RAD51 paralogs assemble into BCDX2 complex to prevent generation of DSBs at stalled replication forks, thereby safeguarding the pre-assembled replisome from the action of nucleases. Strikingly, we showed that RAD51C and XRCC3 in complex with FANCM promote the restart of stalled replication forks in an ATP hydrolysis dependent manner. Moreover, RAD51C R258H mutation that was identified in FA-like disorder abrogates the interaction of RAD51C with FANCM and XRCC3, and prevents fork restart. Thus, assembly of RAD51 paralogs in different complexes prevents nucleolytic degradation of stalled replication forks and promotes restart to maintain genomic integrity. Chapter V: Trans-dichlorooxovandium(IV) complex as a potent photoinducible DNA interstrand crosslinker for targeted cancer therapy Although DNA ICL agents such as MMC, cisplatin and psoralen are known to serve as anticancer drugs, these agents affect normal cells as well. Moreover, tumor resistance to these agents has been reported. We have designed and synthesized a novel photoinducible DNA crosslinking agent (ICL-2) which is a derivative of oxovanadiumterpyridine complex with two chlorides in trans position. We found that ICL-2 can be activated by UV-A and visible light to enable DNA ICLs. ICL-2 efficiently activated FA pathway of ICL repair. Strikingly, photoinduction of ICL-2 induces prolonged activation of cell cycle checkpoint and high degree of cell death in FA pathway defective cells. Moreover, we showed that ICL-2 specifically targets cells that express pathological RAD51C mutants. Our findings suggest that ICL-2 can be potentially used for targeted cancer therapy in patients with gene mutations in FA and HR pathway.

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