Η αδειοδότηση της αντιγραφής αποτελεί μία ιδιαίτερα σημαντική διαδικασία, η οποία λαμβάνει χώρα στις θέσεις έναρξης της αντιγραφής με το σχηματισμό του προ-αντιγραφικού συμπλόκου, που περιλαμβάνει το ORC, το Cdc6, το Cdt1 καθώς και τις MCM 2-7. Η συγκεκριμένη διαδικασία, πραγματοποιείται μία φορά σε κάθε κυτταρικό κύκλο, κατά τη μετάβαση από την M φάση στη G1. Οι παράγοντες που συμμετέχουν υπόκεινται σε εκτεταμένη ρύθμιση προκειμένου να εξασφαλιστεί η απουσία επαναντιγραφής και γονιδιωματικής αστάθειας. Η πρωτεόλυση μορίων που συμμετέχουν στον κυτταρικό κύκλο αποτελεί έναν από τους μηχανισμούς που χρησιμοποιούν τα κύτταρα για τη ρύθμιση των επιπέδων διαφόρων παραγόντων. Το CRL4Cdt2 αποτελεί μία Ε3 λιγάση της ουβικουϊτίνης, η οποία είναι επιφορτισμένη με τη ρύθμιση των επιπέδων του αδειοδοτικού παράγοντα της αντιγραφής Cdt1 κατά την S φάση καθώς και μετά από βλάβες στο γενετικό υλικό. H συγκεκριμένη διαδικασία έχει διαπιστωθεί πως λαμβάνει χώρα σε όλα τα μετάζωα, καθώς και στον σχιζοσακχαρομύκητα. Μεταξύ των υποστρωμάτων του Cdt2 περιλαμβάνονται επίσης το Set8 και το p21. Στην παρούσα διπλωματική εργασία, πραγματοποιήθηκε μελέτη των περιοχών της πρωτεΐνης Cdt2 που είναι υπεύθυνες για τη στρατολόγηση της στην περιοχή της εντοπισμένης βλάβης στο γενετικό υλικό. Η μελέτη πραγματοποιήθηκε σε καρκινικά κύτταρα MCF7 που υπέστησαν εντοπισμένη βλάβη στο γενετικό υλικό με UV-C ακτινοβόληση και χρήση ειδικών πολυκαρβονικών φίλτρων με μικροπόρους. Για τη συγκεκριμένη μελέτη χρησιμοποιήθηκαν φορείς που εκφράζουν το αμινο-τελικό (2-417αα) και το καρβοξυ-τελικό (390-730αα) άκρο της πρωτεΐνης, καθώς και ένα μετάλλαγμα ολόκληρης της πρωτεΐνης όπου 6 SQ θέσεις που εντοπίζονται καρβοξυ-τελικά έχουν μεταλλαχθεί σε αλανίνες και δεν μπορούν να φωσφορυλιωθούν από τις ATM/ATR κινάσες. Μετά τη διεξαγωγή των πειραμάτων συσσώρευσης, διαπιστώθηκε πως στην περιοχή της εντοπισμένης βλάβης στρατολογούνται μόνο το Cdt2 6A(SQ) και Cdt2 (390-730). Ωστόσο, η συσσώρευση τους παρουσιάζεται ελαφρώς ασθενέστερη σε σχέση με την πρωτεΐνη αγρίου τύπου. Η συγκεκριμένη παρατήρηση καταδεικνύει πως στην περίπτωση του Cdt2 6A(SQ) η φωσφορυλίωση στις συγκεκριμένες SQ θέσεις είναι σημαντική για τη συσσώρευση της πρωτεΐνης στην περιοχή της βλάβης. Η στρατολόγηση του Cdt2 (390-730) στην περιοχή της βλάβης, συνιστά μία σημαντική παρατήρηση, η οποία υποδεικνύει ότι η πρωτεΐνη Cdt2 διατηρεί την ικανότητα συσσώρευσης στην περιοχή της βλάβης, παρά την απουσία των αλληλουχιών που βρίσκονται στο αμινο-τελικό άκρο και συμμετέχουν στην αλληλεπίδραση με τα υποστρώματα αλλά και στο σχηματισμό του συμπλόκου της λιγάσης. Προκειμένου να μελετηθεί η κινητική στην περιοχή της εντοπισμένης βλάβης των μεταλλαγμάτων που παρουσιάζουν συσσώρευση, χρησιμοποιήθηκε η τεχνική της επαναφοράς φθορισμού μετά από φωτολεύκανση (FRAP). Τα αποτελέσματα, κατέδειξαν πως τόσο στα κύτταρα χωρίς βλάβη, αλλά και σε αυτά με εντοπισμένη βλάβη υπάρχει παρόμοια κινητική μεταξύ της πρωτεΐνης αγρίου τύπου και των δύο μεταλλαγμάτων. Έτσι, η ύπαρξη μεταλλάξεων σε 6 SQ θέσεις στην περίπτωση του Cdt2 6A(SQ), αλλά και η απώλεια των αμινοτελικών αλληλουχιών στο Cdt2 (390-730) δεν μεταβάλλουν την κινητική στην περιοχή της βλάβης. Τέλος, έγινε μελέτη της συμβολής της φωσφορυλίωσης από τις ATM/ATR κινάσες, στη συσσώρευση στην περιοχή της βλάβης. Γι’ αυτό το λόγο έγινε χρήση της καφεΐνης, που αποτελεί αναστολέα των συγκεκριμένων κινασών. Τα αποτελέσματα, κατέδειξαν πως υπάρχει πολύ ασθενέστερη συσσώρευση στη συγκεκριμένη περίπτωση σε σχέση με την πρωτεΐνη αγρίου τύπου και το Cdt2 6A(SQ). Με αυτό τον τρόπο υποδεικνύεται πως η φωσφορυλίωση σε όλες τις SQ θέσεις του μορίου, είναι σημαντική για τη συσσώρευση του στην περιοχή της βλάβης. / Replication licensing is a crucial process which takes place at the origins of replication. It involves the formation of the pre-replicative complex consisting of ORC, Cdc6, Cdt1 and MCM 2-7. This process takes place during the late M to early G1 transition and happens only once per cell cycle. The tight regulation of the factors that participate in replication licensing prevents rereplication and genomic instability. Proteolysis is a central mechanism of the cell cycle which is important for the regulation of several factors. CRL4Cdt2 is an E3 ubiquitin ligase important for the regulation of the licensing factor Cdt1 during S phase and post DNA damage. It is known that the ubiquitination of Cdt1 by Cdt2 happens in all metazoans and in fission yeast. Set8 and p21 are also substrates of Cdt2. In this study, we investigated the domains of Cdt2 which are important for its recruitment at sites of localized DNA damage. To this end, we employed MCF7 cancer cells which were UV irradiated with polycarbonate filters with micropores. We used plasmids containing N-terminal (2-417aa) and C-terminal (390-730aa) constructs of the protein and a mutant of the entire protein containing alanine substitutions in 6 SQ sites located at the C-terminus of the protein which cannot be phosphorylated by the ATM/ATR kinases. The recruitment experiments indicated that only in the case of Cdt2 (390-730) and Cdt2 6A(SQ) there is recruitment at the site of localized DNA damage. However the recruitment in both cases was weaker compared to the wild type protein. In the case of Cdt2 6A(SQ) this observation indicates the importance of the phosphorylation of the six SQ sites for the recruitment of the protein at the site of damage. Cdt2 (390-730) recruitment indicates that Cdt2 can still get recruited at the site of damage even though it lacks motifs important for the recognition of the substrate and the formation of the ligase complex. In order to study the kinetics of the protein constructs that exhibit recruitment at the site of localized DNA damage we employed Fluorescence Recovery After Photobleaching (FRAP). The results showed that the kinetics of the constructs were similar to the wild type in undamaged cells and in cells with localized damage. This shows that the lack of phosphorylation of 6 SQ sites of Cdt2 6A(SQ) and the loss of the N-terminal motifs of Cdt2 (390-730) are not sufficient to cause any difference in the kinetics at the site of damage. Finally, we wanted to investigate the importance of the phosphorylation by the ATM/ATR kinases for the recruitment of Cdt2 at the sites of localized DNA damage. To this end, we employed the ATM/ATR inhibitor caffeine and we tracked the recruitment at the site of damage. The results showed that the recruitment was weaker compared to the wild type Cdt2 and the Cdt2 6A(SQ). This indicates the importance of the phosphorylation of the 9 SQ sites across the protein for the recruitment at the site of damage.

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Kei Ishii, Masamichi Ishiai, Hiroko Morimoto, Mito Kanatsu-Shinohara, Ohtsura Niwa, Minoru Takata, Takashi Shinohara, The Trp53-Trp53inp1-Tnfrsf10b Pathway Regulates the Radiation Response of Mouse Spermatogonial Stem Cells, Stem Cell Reports, Volume 3, Issue 4, 14 October 2014, Pages 676-689, ISSN 2213-6711 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18685号 / 医博第3957号 / 新制||医||1007(附属図書館) / 31618 / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 藤田 潤, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In response to DNA damage, mammalian cells activate various DNA repair pathways to remove DNA lesions and, meanwhile, halt cell cycle progressions to allow sufficient time for repair. The nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint activation are two major cellular responses to DNA damage induced by UV irradiation. However, how these two processes are coordinated in the response is poorly understood. Here we showed that the essential NER factor XPA (xeroderma pigmentosum group A) underwent nuclear accumulation upon UV irradiation, and strikingly, such an event occurred in an ATR (Ataxia-Telangiectasia mutated and RAD3-related)-dependent manner. Either treatment of cells with ATR kinase inhibitors or transfection of cells with small interfering RNA targeting ATR compromised the UV-induced XPA nuclear translocation. Consistently, the ATR-deficient cells displayed no substantial XPA nuclear translocation while the translocation remained intact in ATM (Ataxia-Telangiectasia mutated)-deficient cells in response to UV irradiation. Moreover, we found that ATR is required for the UV-induced nuclear focus formation of XPA. Taken together, our results suggested that the ATR checkpoint pathway may modulate NER activity through the regulation of XPA redistribution in human cells upon UV irradiation.

Replication Factor A (RFA) is an essential heterotrimeric single-stranded DNA (ssDNA) binding complex, comprised of Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae. RFA is required for DNA replication, repair, recombination, and cell cycle regulation. RFA acts as a sensor of ssDNA, a common intermediate of these processes, and coordinates these processes through recruitment of proteins. For example, during the DNA damage response (DDR), RFA-coated ssDNA is necessary for the recruitment and activation of the sensor kinase Mec1. Additional checkpoint proteins, also recruited by RFA, are necessary for the downstream recruitment and activation of the effector kinase Rad53 that ultimately leads to cell cycle arrest. Thus, RFA acts as a bridge to recruit the proteins required for checkpoint regulation in response to DNA damage. Importantly, cell cycle resumption is contingent on Rad53 deactivation. There are two known scenarios in which Rad53 is deactivated: (1) checkpoint recovery, in which cells resume the cell cycle after DNA repair or (2) checkpoint adaptation, in which cells proceed with the cell cycle despite the continued presence of irreparable DNA damage. Previous work has demonstrated that cells undergoing checkpoint adaptation display late Rfa2 N-terminal (NT) phosphorylation that is correlated with the inactivation (dephosphorylation) of Rad53. Additionally, the use of rfa2 NT mutations consistently demonstrate that a negatively charged NT promotes adaptation in all adaptation-deficient strain backgrounds investigated. Interestingly, Rfa2 NT phosphorylation also occurs early during meiosis. This work demonstrates that: (1) Rfa1-DBD-F participates in protein-protein interactions that are sensitive to DNA damage, (2) Rfa2 phosphorylation increases the DNA damage sensitivity of mutants with deficient DNA damage checkpoints, (3) the Rfa2 NT is required for proper progression through meiosis that appears to be unrelated to RFA functions in replication or DNA repair by homologous recombination (HR), and (4) Rfa2 phosphorylation may regulate Mec1 checkpoint signaling during the DDR to control checkpoint exit and cell cycle resumption. A mechanism is proposed that considers both Rfa1 DBD-F and the Rfa2 NT involvement to initiate HR repair that essentially allows for the continuation of the cell cycle by the delocalization of Mec1.

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No description available.

DNA damage response (DDR) maintains genome integrity through checkpoint activation and lesion repair. While ATM and ATR are essential in DDR, mechanisms regulating their activation remain unclear. BMI1 is a component of the polycomb repressive complex 1 (PRC1), and contributes to PRC1’s E3 ubiquitin (E3-Ub) ligase activity though binding the catalytic subunit RING2. BMI1 binds RING2 through its ring finger (RF) domain. The E3-Ub ligase activity contributes to BMI1-deirved facilitation of the homologous recombination-based repair of DNA double-stranded breaks (DSBs). My research demonstrates that BMI1 reduces ATM and ATR activation during DDR. DSBs and single-strand DNA (ssDNA) lesions respectively activate ATM and ATR. ATM subsequently phosphorylates CHK2 at threonine 68 (CHK2pT68) and induces G2/M arrest. ATR produces CHK1pS345 and S-phase arrest. Both kinases phosphorylate histone H2AX at serine 139 (γH2AX) to prepare for lesion repair. Hydroxyurea initiates DDR via producing ssDNA lesions, and increases ATR activation (phosphorylation of T1989/ATR pT1989), CHK1pS345, γH2AX, and S-phase arrest. These events were significantly reduced and enhanced following the respective BMI1 overexpression and BMI1 knockdown in MCF7 and DU145 cells. BMI1 also displays similar effects towards ATM during DDR induced by etoposide-caused DSBs. Activation of ATM and ATR requires the formation of the ATM-NBS1 and ATR-TOPBP1 complexes. We observed that BMI1 interacted with NBS1 or TOPBP1. Deletion of the RF domain from BMI1 did not affect the associations and also had no effects on BMI1’s activity in reducing ATM activation and ATR-mediated CHK1 pS345. Collectively, our research suggests that BMI1 attenuates ATM and ATR signaling independently of the E3-Ub ligase activity. Genotoxic treatments elicit DDR in cells that are directly exposed and also in cells that are not exposed, a phenomenon known as bystander effect (BE). However, it remains unclear what mediates the BE. Microvesicles are small membrane-enclosed sacks that are shed from donor cells and communicate specific messages to recipient cells. We demonstrated that microvesicles isolated from cells treated with etoposide and ultraviolet induced BE in recipient cells. Neutralization of microvesicles through annexin V reduced the microvesicles-associated BE. / Thesis / Doctor of Philosophy (PhD)

Linear genomes end in characteristic structures consisting of repetitive DNA and proteins: the telomeres. These play two critical roles: on one hand they avoid the of loss of genetic information due to the incomplete replication of the chromosome ends and on the other, they provide capping structures for chromosome termini, differentiating them from double strand breaks. Telomeres contain specialized proteins (the shelterin complex), as well as proteins present elsewhere on the chromosomes (chromatin remodelling, DNA damage repair and response factors). Interestingly, several DNA damage factors are required for proper telomere maintenance, drawing a thin line between telomere protection and their recognition as broken DNA ends. Loss of telomere integrity has severe consequences for the cell, namely it can induce replicative senescence and cellular aging, or it can contribute to tumorigenesis. How telomeres are capped and how they are perceived by the cell when they become dysfunctional is essential for our understanding of the contribution of loss of telomere integrity to aging and disease. In order to unravel new factors involved in telomere maintenance, siRNA screens were performed. The optimization process has confirmed both telomeric foci and telomere dysfunction-induced foci (TIFs) as suitable readouts and the screens performed generated a list of potential candidate genes involved in telomere biology. Although some of the candidate genes tested in this work failed the validation process, other genes deserve further analysis. In addition this work also studied the role of several DNA damage factors at uncapped telomeres. Furthermore, BRCA1, CtIP and EXO1 were found to be critical for the formation of end-to-end fusions generated after TRF2 inactivation. The requirement of this proteins in this process, suggests that not only that not only the classical non-homologous end joining (C-NHEJ) pathway is active at TRF2-depelted telomeres, but emphasises the multiplicity of mechanisms that act to repair dysfunctional telomeres.

The DNA damage response (DDR) is a complex signalling cascade triggered in response to stress, in an attempt to maintain genomic integrity. Components of this pathway, such as ATM-mediated signalling, have been proposed to act as a barrier in the early stages of tumourigenesis. Regions of low oxygen concentrations (hypoxia) occur in most solid tumours and are associated with a poor prognostic outcome. Here, we investigated the DDR induced following hypoxia-induced replication stress in an attempt to decipher the mechanism of ATM activation in response to physiological stresses that do not induce DNA damage. We hypothesized that hypoxia-mediated chromatin changes could impact on ATM signalling. We have characterised H3 methylation in response to hypoxia and found oxygen dependent changes in H3K9me3, including both global and replication fork associated increases in this histone modification. Importantly, we have found that decreases in H3K9me3 result in loss or attenuation of ATM activation. Notably, in a background of replication stress and increased H3K9me3, ATM inhibition or loss leads to accumulation of DNA damage and a significant decrease in replication rates in hypoxia. We propose that when replication stress occurs in the presence of hypoxia-induced chromatin changes, ATM activation is facilitated by the induction of H3K9me3. In this context, we propose a novel and stress specific role for ATM-mediated signalling in maintaining replication and preventing the generation of DNA breaks that may compromise genomic integrity. Moreover, the biological consequences of the hypoxia-induced chromatin context and in particular hypoxia-induced H3K9me3 include the repression of APAK, a negative regulator of p53. Activation of p53 is a key consequence of the hypoxia-induced DDR. Here we found that SETDB1, one of the H3 methyltransferases induced by hypoxia, mediates APAK repression. We propose that H3K9me3 plays a role in regulating APAK expression to allow optimal induction of p53 dependent apoptosis in hypoxic conditions suggesting a further role for H3K9me3 in facilitating DDR signalling in hypoxia. Together, these data suggest that the hypoxic chromatin context is critical for the role of the DDR as a barrier to tumourigenesis and predict that altering the chromatin landscape in combination with DNA damaging therapies would be efficacious in the treatment of hypoxic tumours.