Search

Overexpression in mammalian cells of the error-prone DNA polymerase beta (Pol beta) has been found to increase the spontaneous mutagenesis. Here, we investigated a possible mechanism used by Pol beta to be a genetic instability enhancer: its interference in replicative DNA synthesis, which is normally catalysed by the DNA polymerases alpha, delta and epsilon. By taking advantage of the ability to incorporate efficiently into DNA the chain terminator ddCTP as well as the oxidised nucleotide 8-oxo-dGTP, we show here that purified Pol beta can compete with the replicative DNA polymerases during replication in vitro of duplex DNA when added to human cell extracts. We found that involvement of Pol beta lowers replication fidelity and results in a modified error-specificity. Furthermore, we demonstrated that involvement of Pol beta occurred during synthesis of the lagging strand. These in vitro data provide one possible explanation of how overexpression of the enzyme could perturb the genetic instability in mammalian cells. We discuss these findings within the scope of the up-regulation of Pol beta in many cancer cells.

DNA polymerase beta, one of the most inaccurate DNA synthesizing enzymes, has been shown to confer genetic instability when up-regulated in cells, a situation found in several human cancers. Here, we demonstrated that enhanced activity and expression of this enzyme occur in the human ovarian tumor 2008/C13*5.25 cells, which are resistant to the antitumor agent cisplatin and hypersensitive to 6-thioguanine. We found that translesion synthesis across platinated DNA crosslinks as well as increased incorporation into DNA of 6-thioguanine took place in the 2008/C13*5.25 cells compared to the parental 2008 cells. Such features being molecular signatures of DNA polymerase beta, these findings suggest that deregulation of its expression in cancer cells may contribute to the modulation of the response to antitumor treatments and therefore to tumor progression.

The nucleotide excision repair pathway contributes to genetic stability by removing a wide range of DNA damage through an error-free reaction. When the lesion is located, the altered strand is incised on both sides of the lesion and a damaged oligonucleotide excised. A repair patch is then synthesized and the repaired strand is ligated. It is assumed that only DNA polymerases delta and/or epsilon participate to the repair DNA synthesis step. Using UV and cisplatin-modified DNA templates, we measured in vitro that extracts from cells overexpressing the error-prone DNA polymerase beta exhibited a five- to sixfold increase of the ultimate DNA synthesis activity compared with control extracts and demonstrated the specific involvement of Pol beta in this step. By using a 28 nt gapped, double-stranded DNA substrate mimicking the product of the incision step, we showed that Pol beta is able to catalyze strand displacement downstream of the gap. We discuss these data within the scope of a hypothesis previously presented proposing that excess error-prone Pol beta in cancer cells could perturb the well-defined specific functions of DNA polymerases during error-free DNA transactions.

DNA polymerase beta (Pol beta) is the most inaccurate of the six DNA polymerases found in mammalian cells. In a normal situation, it is expressed at a constant low level and its role is believed to be restricted to repair synthesis in the base excision repair pathway participating to the genome stability. However, excess of Pol beta, found in some human tumors, could confer an increase in spontaneous mutagenesis and result in a highly mutagenic tolerance phenotype toward bifunctional DNA cross-linking anticancer drugs. Here, we present a hypothesis on the mechanisms used by Pol beta to be a genetic instability enhancer through its overexpression. We hypothesize that an excess of Pol beta perturbs the well-defined specific functions of DNA polymerases developed by the cell and propose Pol beta-mediated gap fillings during DNA transactions like repair, replication, or recombination pathways as key processes to introduce illegitimate deoxyribonucleotides or mutagenic base analogs like those produced by intracellular oxidative processes. These mechanisms may predominate during cellular nonproliferative phases in the absence of DNA replication.

DNA polymerase β (pol β) is the most error prone of all known eukaryotic DNA polymerases tested in vitro . Here, we show that cells overexpressing pol β cDNA have acquired a spontaneous mutator phenotype. By measuring the appearance of mutational events using three independent assays, we found that genetic instability increased in the cell lines that overexpressed pol β. In addition, these cells displayed a decreased sensitivity to cancer chemotherapeutic, bifunctional, DNA-damaging agents such as cisplatin, melphalan, and mechlorethamine, resulting in enhanced mutagenesis compared with control cells. By using cell-free extracts and modified DNA substrates, we present data in support of error-prone translesion replication as one of the key determinants of tolerance phenotype. These results have implications for the potential role of pol β overexpression in cancer predisposition and tumor progression during chemotherapy.

DNA replication is an asymmetric process involving concurrent DNA synthesis on leading and lagging strands. Leading strand synthesis proceeds concomitantly with fork opening, whereas synthesis of the lagging strand essentially takes place on a single-stranded template. The effect of this duality on DNA damage processing by the cellular replication machinery was tested using eukaryotic cell extracts and model DNA substrates containing site-specific DNA adducts formed by the anticancer drug cisplatin or by the carcinogen N -2-acetylaminofluorene. Bypass of both lesions was observed only with fork-like substrates, whereas complete inhibition of DNA synthesis occurred on damaged single-stranded DNA substrates. These results suggest a role for additional accessory factors that permit DNA polymerases to bypass lesions when present in fork-like DNA.

Cisplatin [cis-diamminedichloroplatinum II] is widely used in the treatment of a broad range of tumors. A number of biological and biochemical results indicate that the reaction of cisplatin with DNA is responsible for the cytotoxic action of the drug. However, cisplatin can induce mutagenesis and may be carcinogenic in humans. Error prone replication of damaged DNA must be considered as a possible mechanism of mutagenesis. In this short review, we present data indicating that DNA containing cisplatin lesions can be replicated by prokaryotes and eukaryotes in a mutagenic fashion.

The conventional clinical staging classification is not sufficient to predict the survival of patients who suffer from early lung cancer. Additional prognostic factors are then needed to better forecast their outcome. Since perturbation of the genome replication i.e. the so-called “replicative stress” is admitted to contribute to neoplasia from its early stages, we hypothesized that genes involved in such process may therefore represent a still under-explored source of such biomarkers. We specifically assessed in primary tumors and adjacent normal tissues from a series of 93 patients suffering from early- or mid-stage non-small cell lung adenocarcinoma the expression (RT-qPCR) of 77 “DNA replication” genes involved in either initiation/licensing at the 50,000 replication origins dispersed along the genome, elongation of DNA chains onto damaged or undamaged DNA, maintenance of stalled DNA replication forks or intra-S phase DNA damage processing and signaling. A 4-gene signature separated patients to high-risk and low-risk subgroups with significantly different survival. This prognostic effect was independent on age, sex, treatment, stage classification and expression of proliferation markers. We propose that a “cancer replisome” signature could be a predictor of the cancer survival and might also help understanding the molecular mechanisms underlying tumor progression in lung cancer patients.

For many years, we have advanced the hypothesis that cancers exhibit a mutator phenotype. Recent support for this concept was gained by the increased frequency of random single-base substitutions measured in human tumors as well as by the large numbers of clonal mutations detected by DNA sequencing in a variety of human cancers. While these findings indicate that cancer cells accumulate large numbers of mutations, it remains unclear what types of alterations in the fidelity of DNA synthesis contribute most to the evolution of a tumor. In order to delineate the types of mutations that have the greatest impact on accelerating tumorigenesis, we studied the consequences of altered accuracy of synthesis by mutant DNA polymerases. DNA polymerase δ (Pol δ) is responsible for replication of the lagging DNA strand during replication and functions in multiple DNA repair pathways. Based on homology to substitutions known to induce error-prone synthesis in other DNA polymerases, we constructed a series of single base substitutions at position L606 in the catalytic domain of human Pol δ. We found that Pol δ L606G introduces a high frequency of point mutations into DNA in vitro, but heterozygous mice expressing L606G do not exhibit a cancer phenotype. In contrast, Pol δ L606K synthesizes DNA in vitro with high accuracy; yet heterozygous mice expressing this mutant exhibit enhanced rates of tumor progression. Compared to either the wild type or L606G, the L606K mutant is defective in lesion bypass; cells expressing this mutant exhibit a decrease in the rate of fork progression. In addition, L606K cells exhibit an increase in spontaneous chromosomal aberrations, and comparative genomic hybridization analysis reveals an increased frequency of random deletions in tumors from mice expressing Pol δ L606K. These results indicate that mutations in a major replicating DNA polymerase can result in random deletions throughout the genome that have the potential to affect tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 241.

The use of substrates containing well defined adducts at precise sites, is required to perform a careful analysis of the toxic and mutagenic potential of a lesion. As a first step in this direction the octamer 5'-d(CCGGCGGT), containing the sequence of the codons 12 d(GGC) and 13 d(GGT) of the human H-ras gene, was reacted with the antitumoral drug cis-diamminedichloroplatinum(II). The platinated products have been purified by HPLC. A first set of experiments, including enzymatic digestions with nuclease P1 followed by alkaline phosphatase and acid-catalysed hydrolysis, allowed us to determine which bases were engaged in the cis-DDP lesions. Our results indicate that only guanine residues were chelated with cisplatin to yield bifunctional adducts. Furthermore, by performing enzymatic digestions with phosphodiesterases, we have located the adducts with respect to the 5' end of the octamer. Among the purified and characterized platinated oligonucleotides, three present a particular interest, since we have shown here that the cis-d(GpG) adduct is precisely situated either at the d(GGC) or at the d(GGT) or at both sites of their sequence.

We have compared the mode of fixation in vitro of the antitumor drug cis -diamminedichloroplatinum (II) ( cis -DDP) to single-stranded M13mp10DNA either in the presence or absence of the Escherichia coli single-stranded binding protein (SSB). Platinum binding sites have been identified by taking advantage of their capacity to inhibit DNA replication of primed M13 DNA catalysed by E . coli DNA polymerase I large fragment. We report here that the presence of SSB increases the number of platinum-DNA lesions and alters their distribution. We also present evidence that SSB allows cis -DDP to bind to DNA sequences otherwise less accessible.

Cytotoxicity and antitumor activity of platinum complexes are often sensitive to structural features of the platinum compound such as its isomerism, the number of leaving groups, and the nature of the stable ligand. Whereas cis-[Ptcl2(NH3)2] (cis-DDP) is an antitumor drug which is active against human and murine cancers, monofunctional compounds such as [PtCl(dien)]Cl and the trans isomer, trans-DDP, have no antitumor activity (Braddock et al., 1975; Macquet and Butour, 1983). During the reaction with DNA, the N(7) atoms of purine bases substitute the labile Cl ligands while the non-leaving group, NH3, remains bound to the metal. Non-leaving groups play an important role in modulating the antitumor activity of platinum compounds. For example, cell lines resistant to cis-DDP are not cross-resistant to platinum complexes with 1,2-diaminocyclohexane (DACH) (Burchenal et al., 1979). Platinum complexes with alkylamine and pyridine ligands are less antitumor than cis-DDP or inactive (Braddock et al., 1975; Meischen et al., 1976; Farrell et al., 1989).

Reaction of cis-diamminedichloroplatinum (II) with single-stranded M13 phage DNA in vitro produced monofunctional platinum-DNA adducts on guanine and bifunctional lesions with either two guanine bases (GG) or one adenine and one guanine (AG). When DNA containing a majority of monofunctional platinum-DNA lesions was dialyzed against 10 mM NaCIO4 at 37 degrees C, conversion of monoadducts to bifunctional lesions was observed. We examined the effect of post-treatment formation of bifunctional lesions on DNA synthesis by Escherichia coli DNA polymerase I and highly purified eukaryotic DNA polymerase alpha from Drosophila melanogaster and calf thymus. Arrest sites on the platinated template were determined by polyacrylamide gel electrophoresis. Monofunctional lesions did not appear to block DNA synthesis. Inhibition of replication increased as bifunctional platinum-DNA lesions formed during post-treatment incubation; GG adducts inhibited replication more than AG. These results suggest that bifunctional GG platinum-DNA adducts may be the major toxic damage of cisplatin.

Oxidative stress has been proposed to be one of the major causes leading to the accumulation of mutation that is associated with the initiation and progression of cancers. Elevated expression of DNA polymerase beta, an event found in many human tumors, has been shown to generate a mutator phenotype. Here, we demonstrated that overexpression of DNA polymerase beta strengthens the mutagenicity of oxidative damages, concomitantly with a higher cellular sensitivity and increased apoptosis. Deregulated expression of DNA polymerase beta could represent a predisposition factor for mutagenic effects of oxidative stress and thus have implication in the generation and/or evolution of cancer.

The physiological function of the human DNA polymerase θ (pol θ) is still unclear despite its in vitro translesion synthesis capacity during DNA damage repair process. However this DNA polymerase is always present along the cell cycle in the absence of replication stress and DNA damage. Is there a different molecular function? We present the genomic data of replication timing in depleted pol θ cells (GSE49693) and in cells overexpressing pol θ (GSE53070) indicating that Pol θ holds a novel role in the absence of external stress as a critical determinant of the replication timing program in human cells.

DNA polymerase β (Pol β) is an error-prone repair DNA polymerase that has been shown to create genetic instability and tumorigenesis when overexpressed by only 2-fold in cells, suggesting that a rigorous regulation of its expression may be essential in vivo. To address this question, we have generated mice which express a transgene (Tg) bearing the Pol β cDNA under the control of the ubiquitous promoter of the mouse H-2K gene from the major histocompatibility complex. These mice express the Tg only in thymus, an organ which normally contains the most abundant endogenous Pol β mRNA and protein, supporting the idea of a tight regulation of Pol β in vivo. Furthermore, we found no tumor incidence, suggesting that the single Pol β overexpression event is not sufficient to initiate tumorigenesis in vivo.

DNA duplication in S phase starts from thousands of initiation sites called DNA replication origins. These replication origins are set in G1 as pre-replication complexes (pre-RC) and fired in S phase following a spatio-temporal program of activation. This program determines which origins will be fired and when. Indeed, all the origins are not fired in the same time and we can distinguish early, middle and late replication origins. This temporal regulation is called "replication timing" and is determined at the "timing decision point" (TDP) in early G1. It's a robust program, which participates to the definition of cell identity, in term of differentiation state or cell type. However, the precise molecular mechanisms involved are poorly understood. Defective timing program has been evidenced in pathological contexts, in particular in cancers, but the mechanisms of this deregulation remain unclear. In the first part of my PhD, I contributed to the discovery of a new regulator of the origin timing program: the specialized DNA polymerase Theta (Pol Theta). Pol Theta is loaded onto chromatin in early G1, coimmunoprecipitates with pre-RC components and modulates the recruitment of Mcm helicases at TDP. Moreover, depletion or overexpression of Pol Theta modifies the timing of replication at a fraction of chromosomal domains. The second part of my work aimed at exploring the mechanisms that regulates replication timing after a replicative stress. I identified a totally new transgenerational adaptive mechanism of DNA replication timing regulation: the modification of the timing of origin activation at chromosomal domains that have suffered from a replicative stress during the previous cell cycle. Daughter cells from a cell that has experienced replication stress at particular domains (late replicating domains, AT rich so they can form structured DNA, and poor in origin density) shows advanced origin activation within these regions. This new biological process in response to replicative stress could be of particular interest in the context of cancer since, tumor cells are characterized by high level of intrinsic chronic replicative stress. This new mechanism may favor cancer cell survival despite replication stress, particularly upon treatments with anti-tumor agents that target DNA., La duplication de l'ADN au cours de la phase S est initiée à partir de l'activation de plusieurs dizaines de milliers d'origines de réplication. La mise en place des origines a lieu au cours de la phase G1 sous la forme de complexe de pré-réplication (pré-RC) et leur activation est orchestrée par un programme spatio-temporel. La régulation spatiale détermine les origines qui seront activées et la régulation temporelle, ou timing de réplication, détermine le moment de leur activation. En effet, toutes ces origines ne sont pas activées en même temps durant la phase S : certaines origines seront activées en début de phase S, d'autre en milieu, ou d'autre à la fin. Ce programme est établi en tout début de phase G1, au " point de décision du timing ". C'est un programme très robuste qui signe l'identité d'une cellule, son état de différenciation et le type cellulaire à laquelle elle appartient. Il a aussi été montré qu'il est altéré dans des situations pathologiques, en particulier le cancer, sans qu'on ne comprenne très bien les raisons mécanistiques. De manière générale, les mécanismes moléculaires qui régulent le timing de réplication sont méconnus. Le premier volet de ma thèse a permis l'identification d'un nouveau régulateur du timing de réplication : il s'agit de l'ADN polymérase spécialisée Thêta. Recrutée à la chromatine très tôt en phase G1, elle interagit avec des composants du pré-RC, et régule le recrutement des hélicases réplicatives à la chromatine. Enfin, sa déplétion ou sa surexpression entraîne une modification du timing de réplication à l'échelle du génome. Dans la deuxième partie de ma thèse, j'ai exploré les mécanismes qui régulent ce programme temporel d'activation des origines suite à un stress réplicatif. J'ai identifié un mécanisme de régulation transgénérationnel inédit : la modification du timing de réplication de domaines chromosomiques ayant subi un stress réplicatif au cycle cellulaire précédent. Des cellules-filles issues d'une cellule ayant subi des problèmes de réplication dans des domaines fragiles (riches en AT, et donc potentiellement structurés, et pauvres en origines) présentent un timing plus précoce de l'activation des origines au niveau de ces domaines. Ce nouveau processus biologique d'adaptation est particulièrement intéressant dans un contexte tumoral de haut stress réplicatif chronique car ce pourrait être un moyen pour la cellule tumorale de survivre à son propre stress réplicatif mais aussi aux thérapies antitumorales qui sont nombreuses à cibler la réplication de l'ADN.

DNA duplication in S phase starts from thousands of initiation sites called DNA replication origins. These replication origins are set in G1 as pre-replication complexes (pre-RC) and fired in S phase following a spatio-temporal program of activation. This program determines which origins will be fired and when. Indeed, all the origins are not fired in the same time and we can distinguish early, middle and late replication origins. This temporal regulation is called "replication timing" and is determined at the "timing decision point" (TDP) in early G1. It's a robust program, which participates to the definition of cell identity, in term of differentiation state or cell type. However, the precise molecular mechanisms involved are poorly understood. Defective timing program has been evidenced in pathological contexts, in particular in cancers, but the mechanisms of this deregulation remain unclear. In the first part of my PhD, I contributed to the discovery of a new regulator of the origin timing program: the specialized DNA polymerase Theta (Pol Theta). Pol Theta is loaded onto chromatin in early G1, coimmunoprecipitates with pre-RC components and modulates the recruitment of Mcm helicases at TDP. Moreover, depletion or overexpression of Pol Theta modifies the timing of replication at a fraction of chromosomal domains. The second part of my work aimed at exploring the mechanisms that regulates replication timing after a replicative stress. I identified a totally new transgenerational adaptive mechanism of DNA replication timing regulation: the modification of the timing of origin activation at chromosomal domains that have suffered from a replicative stress during the previous cell cycle. Daughter cells from a cell that has experienced replication stress at particular domains (late replicating domains, AT rich so they can form structured DNA, and poor in origin density) shows advanced origin activation within these regions. This new biological process in response to replicative stress could be of particular interest in the context of cancer since, tumor cells are characterized by high level of intrinsic chronic replicative stress. This new mechanism may favor cancer cell survival despite replication stress, particularly upon treatments with anti-tumor agents that target DNA.; La duplication de l'ADN au cours de la phase S est initiée à partir de l'activation de plusieurs dizaines de milliers d'origines de réplication. La mise en place des origines a lieu au cours de la phase G1 sous la forme de complexe de pré-réplication (pré-RC) et leur activation est orchestrée par un programme spatio-temporel. La régulation spatiale détermine les origines qui seront activées et la régulation temporelle, ou timing de réplication, détermine le moment de leur activation. En effet, toutes ces origines ne sont pas activées en même temps durant la phase S : certaines origines seront activées en début de phase S, d'autre en milieu, ou d'autre à la fin. Ce programme est établi en tout début de phase G1, au " point de décision du timing ". C'est un programme très robuste qui signe l'identité d'une cellule, son état de différenciation et le type cellulaire à laquelle elle appartient. Il a aussi été montré qu'il est altéré dans des situations pathologiques, en particulier le cancer, sans qu'on ne comprenne très bien les raisons mécanistiques. De manière générale, les mécanismes moléculaires qui régulent le timing de réplication sont méconnus. Le premier volet de ma thèse a permis l'identification d'un nouveau régulateur du timing de réplication : il s'agit de l'ADN polymérase spécialisée Thêta. Recrutée à la chromatine très tôt en phase G1, elle interagit avec des composants du pré-RC, et régule le recrutement des hélicases réplicatives à la chromatine. Enfin, sa déplétion ou sa surexpression entraîne une modification du timing de réplication à l'échelle du génome. Dans la deuxième partie de ma thèse, j'ai exploré les mécanismes qui régulent ce programme temporel d'activation des origines suite à un stress réplicatif. J'ai identifié un mécanisme de régulation transgénérationnel inédit : la modification du timing de réplication de domaines chromosomiques ayant subi un stress réplicatif au cycle cellulaire précédent. Des cellules-filles issues d'une cellule ayant subi des problèmes de réplication dans des domaines fragiles (riches en AT, et donc potentiellement structurés, et pauvres en origines) présentent un timing plus précoce de l'activation des origines au niveau de ces domaines. Ce nouveau processus biologique d'adaptation est particulièrement intéressant dans un contexte tumoral de haut stress réplicatif chronique car ce pourrait être un moyen pour la cellule tumorale de survivre à son propre stress réplicatif mais aussi aux thérapies antitumorales qui sont nombreuses à cibler la réplication de l'ADN.

DNA replication is a major event for cells which allow the faithful duplication of genetic material. It is a critical step of cell cycle, because replication forks encounters frequently naturals barriers (non B-DNA structures), exogenous barriers (chemicals agents), or endogenous barriers (oxydatives lesions). These different barriers can be at the origin of chromosomes breaks and lead to genetic instability. To overcome the stalled forks, cells have evolved two major mechanisms: the induction of ATR replication checkpoint pathway and the recruitment of specialized DNA polymerase to perform the translesion synthesis. This two pathways are essential to maintain genomic stability. Human DNA polymerase Kappa (pol Kappa), the most conserved specialized DNA polymerase, is best known to participate to translesion synthesis. Recently, we have shown that pol kappa has a crucial role in the S-phase checkpoint activation. Indeed, pol Kappa is implicated in the synthesis of short DNA intermediates at the stalled forks, facilitating the recruitment of 9-1-1 clamp, and is required for an optimal phosphorylation of Chk1, the main effector of the S-phase checkpoint. Durant my PhD thesis, I explored the molecular mechanisms underlying this newly identified role. We have constructed several pol kappa mutants, and we have observed that for the mutation in the PCNA binding domain impeded pol kappa to form foci in response to replication stress. We also showed the requirement of this domain for pol Kappa recruitment on chromatin. By different experimental approaches, we have described a complex in which pol Kappa interacts with Rad9 and Chk1, two proteins required for the S-phase checkpoint activation. The Chk1 phosphorylation defect observed in absence of Kappa could also be the consequence of the Chk1 protein level decreased in the nucleus, meaning a potential common regulation between pol Kappa and Chk1. Based on this observation, we have studied how pol Kappa is regulated upon a replication stress and like Chk1, pol Kappa seems to be regulated by ubiquitination. We focused our attention on USP7 an ubiquitin hydrolase known to regulate Chk1. We have demonstrated an interaction between pol Kappa and USP7, which is stimulated after replication stress. Moreover, USP7 depletion leads to a decrease of pol Kappa level in the nucleus, suggesting that de-ubiquination of pol Kappa could to be a prerequisite for its checkpoint function and its stability.; La réplication de l'ADN est un évènement majeur pour la cellule car elle permet la duplication fidèle du matériel génétique. Il s'agit d'une étape critique du cycle cellulaire, car les fourches de réplication rencontrent fréquemment des barrières naturelles ou des lésions d'origine endogènes (lésions oxydatives) ou exogènes (agents physiques ou chimiques), sources de cassures chromosomiques et donc d'instabilité génétique. Une des réponses à ces fourches bloquées est l'activation du point de contrôle (checkpoint) de la phase S du cycle cellulaire. Nous avons montré que l'ADN polymérase Kappa (pol Kappa), polymérase dite translesionnelle en raison de ses capacité à franchir des lésions sur l'ADN, s'avère être aussi un acteur du point de contrôle de phase S. En effet, la déplétion de pol Kappa par ARN interférence dans différentes lignées cellulaires ou par immunodépletion d'un extrait de Xénope, entraîne un défaut de phosphorylation de Chk1. Pol Kappa est impliquée dans la synthèse de brins naissant d'ADN au niveau des fourches bloquées, ce qui facilite le recrutement du complexe 9-1-1 composé des protéines Rad9, Rad1 et Hus1et permet alors, une activation correcte du checkpoint de phase S. Afin de décrypter le rôle de pol kappa, nous avons construits différents mutants et nous avons analysé leur capacité à former des foyers, à être recrutés à la chromatine et à interagir avec différents partenaires dans des conditions d'activation du point de contrôle de phase S. Nous avons pu constater que le mutant du domaine d'interaction à PCNA était incapable de former des foci foyers ?. Nous avons ensuite observé, qu'en condition de stress réplicatif, pol Kappa était recruté à la chromatine grâce à son domaine d'interaction à PCNA et par différentes approches biochimiques, nous avons pu voir que pol kappa interagissait avec Rad9 et Chk1. Nous avons également mis en évidence que le défaut d'activation de Chk1 en l'absence de pol kappa reflétait d'une diminution de son taux dans le noyau, suggérant une régulation commune entre Chk1 et pol Kappa. En effet, nous avons observé que pol Kappa, comme Chk1, était régulés par l'ubiquitine hydrolase USP7. En effet, l'interaction entre pol Kappa et USP7 est augmentée en condition de stress. Nous avons pu voir, qu'à l'instar de Chk1, l'absence de USP7 entrainait une baisse du niveau de pol kappa dans le noyau. Ainsi nous proposons qu'en réponse à un stress réplicatif, pol Kappa et Chk1 soient stabilisés via leur dé-ubiquitination par USP7, permettant leur recrutement à la chromatine et une activation correcte du checkpoint de phase S. Parallèlement à ces travaux, des publications récentes montrent une implication possible de pol Kappa au niveau des séquences répétées. Nous avons pu mettre en évidence une interaction entre pol Kappa et Cenpb, protéine centromérique reconnaissant une séquence de 17 paires de bases dans l'ADN a-satellite. Ces résultats préliminaires suggèrent que le rôle de pol Kappa dans le checkpoint de phase S s'adresse notamment aux régions d'hétérochromatine. L'ensemble des résultats obtenus montre l'importance de pol Kappa dans le maintien de la stabilité génomique, par son rôle dans le checkpoint de phase S, et par son implication dans la régulation de Chk1 en condition de stress réplicatif.

International audience

DNA replication is a major event for cells which allow the faithful duplication of genetic material. It is a critical step of cell cycle, because replication forks encounters frequently naturals barriers (non B-DNA structures), exogenous barriers (chemicals agents), or endogenous barriers (oxydatives lesions). These different barriers can be at the origin of chromosomes breaks and lead to genetic instability. To overcome the stalled forks, cells have evolved two major mechanisms: the induction of ATR replication checkpoint pathway and the recruitment of specialized DNA polymerase to perform the translesion synthesis. This two pathways are essential to maintain genomic stability. Human DNA polymerase Kappa (pol Kappa), the most conserved specialized DNA polymerase, is best known to participate to translesion synthesis. Recently, we have shown that pol kappa has a crucial role in the S-phase checkpoint activation. Indeed, pol Kappa is implicated in the synthesis of short DNA intermediates at the stalled forks, facilitating the recruitment of 9-1-1 clamp, and is required for an optimal phosphorylation of Chk1, the main effector of the S-phase checkpoint. Durant my PhD thesis, I explored the molecular mechanisms underlying this newly identified role. We have constructed several pol kappa mutants, and we have observed that for the mutation in the PCNA binding domain impeded pol kappa to form foci in response to replication stress. We also showed the requirement of this domain for pol Kappa recruitment on chromatin. By different experimental approaches, we have described a complex in which pol Kappa interacts with Rad9 and Chk1, two proteins required for the S-phase checkpoint activation. The Chk1 phosphorylation defect observed in absence of Kappa could also be the consequence of the Chk1 protein level decreased in the nucleus, meaning a potential common regulation between pol Kappa and Chk1. Based on this observation, we have studied how pol Kappa is regulated upon a replication stress and like Chk1, pol Kappa seems to be regulated by ubiquitination. We focused our attention on USP7 an ubiquitin hydrolase known to regulate Chk1. We have demonstrated an interaction between pol Kappa and USP7, which is stimulated after replication stress. Moreover, USP7 depletion leads to a decrease of pol Kappa level in the nucleus, suggesting that de-ubiquination of pol Kappa could to be a prerequisite for its checkpoint function and its stability., La réplication de l'ADN est un évènement majeur pour la cellule car elle permet la duplication fidèle du matériel génétique. Il s'agit d'une étape critique du cycle cellulaire, car les fourches de réplication rencontrent fréquemment des barrières naturelles ou des lésions d'origine endogènes (lésions oxydatives) ou exogènes (agents physiques ou chimiques), sources de cassures chromosomiques et donc d'instabilité génétique. Une des réponses à ces fourches bloquées est l'activation du point de contrôle (checkpoint) de la phase S du cycle cellulaire. Nous avons montré que l'ADN polymérase Kappa (pol Kappa), polymérase dite translesionnelle en raison de ses capacité à franchir des lésions sur l'ADN, s'avère être aussi un acteur du point de contrôle de phase S. En effet, la déplétion de pol Kappa par ARN interférence dans différentes lignées cellulaires ou par immunodépletion d'un extrait de Xénope, entraîne un défaut de phosphorylation de Chk1. Pol Kappa est impliquée dans la synthèse de brins naissant d'ADN au niveau des fourches bloquées, ce qui facilite le recrutement du complexe 9-1-1 composé des protéines Rad9, Rad1 et Hus1et permet alors, une activation correcte du checkpoint de phase S. Afin de décrypter le rôle de pol kappa, nous avons construits différents mutants et nous avons analysé leur capacité à former des foyers, à être recrutés à la chromatine et à interagir avec différents partenaires dans des conditions d'activation du point de contrôle de phase S. Nous avons pu constater que le mutant du domaine d'interaction à PCNA était incapable de former des foci foyers ?. Nous avons ensuite observé, qu'en condition de stress réplicatif, pol Kappa était recruté à la chromatine grâce à son domaine d'interaction à PCNA et par différentes approches biochimiques, nous avons pu voir que pol kappa interagissait avec Rad9 et Chk1. Nous avons également mis en évidence que le défaut d'activation de Chk1 en l'absence de pol kappa reflétait d'une diminution de son taux dans le noyau, suggérant une régulation commune entre Chk1 et pol Kappa. En effet, nous avons observé que pol Kappa, comme Chk1, était régulés par l'ubiquitine hydrolase USP7. En effet, l'interaction entre pol Kappa et USP7 est augmentée en condition de stress. Nous avons pu voir, qu'à l'instar de Chk1, l'absence de USP7 entrainait une baisse du niveau de pol kappa dans le noyau. Ainsi nous proposons qu'en réponse à un stress réplicatif, pol Kappa et Chk1 soient stabilisés via leur dé-ubiquitination par USP7, permettant leur recrutement à la chromatine et une activation correcte du checkpoint de phase S. Parallèlement à ces travaux, des publications récentes montrent une implication possible de pol Kappa au niveau des séquences répétées. Nous avons pu mettre en évidence une interaction entre pol Kappa et Cenpb, protéine centromérique reconnaissant une séquence de 17 paires de bases dans l'ADN a-satellite. Ces résultats préliminaires suggèrent que le rôle de pol Kappa dans le checkpoint de phase S s'adresse notamment aux régions d'hétérochromatine. L'ensemble des résultats obtenus montre l'importance de pol Kappa dans le maintien de la stabilité génomique, par son rôle dans le checkpoint de phase S, et par son implication dans la régulation de Chk1 en condition de stress réplicatif.