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DNA lesions in S phase threaten genome stability. The DNA damage tolerance (DDT) pathways overcome these obstacles and allow completion of DNA synthesis by the use of specialised translesion (TLS) DNA polymerases or through recombination-related processes. However, how these mechanisms coordinate with each other and with bulk replication remains elusive. To address these issues, we monitored the variation of replication intermediate architecture in response to ultraviolet irradiation using transmission electron microscopy. We show that the TLS polymerase η, able to accurately bypass the major UV lesion and mutated in the skin cancer-prone xeroderma pigmentosum variant (XPV) syndrome, acts at the replication fork to resolve uncoupling and prevent post-replicative gap accumulation. Repriming occurs as a compensatory mechanism when this on-the-fly mechanism cannot operate, and is therefore predominant in XPV cells. Interestingly, our data support a recombination-independent function of RAD51 at the replication fork to sustain repriming. Finally, we provide evidence for the post-replicative commitment of recombination in gap repair and for pioneering observations of in vivo recombination intermediates. Altogether, we propose a chronology of UV damage tolerance in human cells that highlights the key role of polη in shaping this response and ensuring the continuity of DNA synthesis.

Genetic instability is a hallmark of both cancer and aging. Homologous recombination (HR) is a prominent DNA repair pathway maintaining genomic integrity. Mutations in many HR genes lead to cancer predisposition. Paradoxically, the consequences of mutations in the pivotal HR player, RAD51, on cancer development remain puzzling. Moreover, in contrast with other HR genes, RAD51 mouse models are not available to experimentally address the role of RAD51 on aging and carcinogenesis, in vivo. Here, we engineered a mouse model with an inducible dominant negative form of RAD51 (SMRad51) that suppresses RAD51-mediated HR without stimulating alternative non-conservative repair pathways. We found that, in vivo expression of SMRad51 did not trigger tumorigenesis, but instead induced premature aging. We propose that these in vivo phenotypes result from the exhaustion of proliferating progenitors submitted to chronic endogenous replication stress resulting from RAD51-mediated HR suppression. Our data underline the importance of the RAD51 activity for progenitors homeostasis, preventing aging, and more generally for the balance between cancer and aging.

Tumors with microsatellite instability (MSI) represent a paradigm for the success of immune checkpoint inhibitor (ICI)-based immunotherapy, particularly in patients with metastatic colorectal cancer (mCRC). To date however, tools for predicting efficacy of these new therapies are lacking. Here we combined high-throughput DNA and RNA sequencing of tumors from 117 patients with MSI mCRC treated with anti-PD-1 +/- anti-CTLA-4 and enrolled into two cohorts, i.e., the NIPICOL clinical trial (NCT03350126) and the ImmunoMSI prospective cohort that were used as discovery and validation cohorts in this ancillary study, respectively. All analyses based on previously suggested DNA/RNA biomarkers of resistance failed to identify robust predictors of treatment response in patients, e.g., the level of MSI, mutational burden, the presence of specific somatic DNA variants as assessed by exome-sequencing and the activity of canonical signaling pathways or the presence of specific cellular contingents within the tumor bulk as estimated by RNA-sequencing. By contrast, resistance to ICI was found to depend both on a small subset of somatic DNA variants located in microsatellite-containing genes with very diverse biological functions and the expression of a stromal-oriented RNA component. Testing of these predictors in 5 large additional independent retrospective and prospective cohorts (IDEA and MOSAIC clinical trials) regrouping 446 patients with nonmetastatic or metastatic MSI CRC untreated with ICI allowed us to validate the specificity of the predictive nature of these indicators regarding ICI-treated MSI mCRC patients. The use of these DNA/RNA predictors will help to refine the ICI-based precision therapy of patients with metastatic MSI mCRC.

The DNA polymerase zeta (Polζ) plays a critical role in bypassing DNA damage. REV3L, the catalytic subunit of Polζ, is also essential in mouse embryonic development and cell proliferation for reasons that remain incompletely understood. In this study, we reveal that REV3L protein interacts with heterochromatin components including repressive histone marks and localizes in pericentromeric regions through direct interaction with HP1 dimer. We demonstrate that Polζ/REV3L ensures progression of replication forks through difficult‐to‐replicate pericentromeric heterochromatin, thereby preventing spontaneous chromosome break formation. We also find that Rev3l‐deficient cells are compromised in the repair of heterochromatin‐associated double‐stranded breaks, eliciting deletions in late‐replicating regions. Lack of REV3L leads to further consequences that may be ascribed to heterochromatin replication and repair‐associated functions of Polζ, with a disruption of the temporal replication program at specific loci. This is correlated with changes in epigenetic landscape and transcriptional control of developmentally regulated genes. These results reveal a new function of Polζ in preventing chromosome instability during replication of heterochromatic regions., HP1‐mediated targeting of REVL3, the catalytic subunit of translesion synthesis Polζ, influences replication fork progression and epigenetic and transcriptional landscapes of developmentally‐regulated genes.

DNA lesions in S phase threaten genome stability. The DNA damage tolerance (DDT) pathways overcome these obstacles and allow completion of DNA synthesis by the use of specialised translesion (TLS) DNA polymerases or through recombination-related processes. However, how these mechanisms coordinate with each other and with bulk replication remain elusive. To address these issues, we monitored the variation of replication intermediate architecture in response to ultraviolet irradiation using transmission electron microscopy. We show that the TLS polymerase η, able to accurately bypass the major UV lesion and mutated in the skin cancer-prone xeroderma pigmentosum variant (XPV) syndrome, acts at the replication fork to resolve uncoupling and prevent post-replicative gap accumulation. Repriming occurs as a compensatory mechanism when this on-the-fly mechanism cannot operate, and is therefore predominant in XPV cells. Interestingly, our data support a recombination-independent function of RAD51 at the replication fork to sustain repriming. Finally, we provide evidence for the post-replicative commitment of recombination in gap repair and for pioneering observations of in vivo recombination intermediates. Altogether, we propose a chronology of UV damage tolerance in human cells that highlights the key role of polη in shaping this response and ensuring the continuity of DNA synthesis.

Immune checkpoint inhibitors have demonstrated unprecedented clinical activity in a wide range of cancers. Significant therapeutic responses have recently been observed in patients presenting mismatch repair-deficient (MMRD) tumours. MMRD cancers exhibit a remarkably high rate of mutations, which can result in the formation of neoantigens, hypothesised to enhance the antitumour immune response. In addition to MMRD tumours, cancers mutated in the exonuclease domain of the catalytic subunit of the DNA polymerase epsilon (POLE) also exhibit an ultramutated genome and are thus likely to benefit from immunotherapy. In this review, we provide an overview of recent data on hypermutated tumours, including MMRD and POLE-mutated cancers, with a focus on their distinctive clinicopathological and molecular characteristics as well as their immune environment. We also discuss the emergence of immune therapy to treat these hypermutated cancers, and we comment on the recent Food and Drug Administration approval of an immune checkpoint inhibitor, the programmed cell death 1 antibody (pembrolizumab, Keytruda), for the treatment of patients with metastatic MMRD cancers regardless of the tumour type. This breakthrough represents a turning point in the management of these hypermutated tumours and paves the way for broader strategies in immunoprecision medicine.

Oncogenes trigger replicative stress that can lead to genetic instability, which participates in cancer progression. Thus, determining how cells cope with replicative stress can help our understanding of oncogenesis and lead to the identification of new antitumor treatment targets. We previously showed that constitutive overexpression of the oncogenic transcription factor Spi1/PU.1 leads to pre-leukemic cells that have a shortened S phase duration with an increased replication fork speed and increased mutability in the absence of DNA breaks. Here, we demonstrate that the S phase checkpoint protein CHK1 is maintained in a low phosphorylation state in Spi1/PU.1-overexpressing cells and provide evidence that this is not due to negative control of its primary kinase ATR. Notably, we found that the expression of the CHK1 phosphatase PP1α is increased in Spi1/PU.1-overexpressing cells. By exogenously modulating its activity, we demonstrate that PP1α is required to maintain CHK1 in a dephosphorylated state and, more importantly, that it is responsible for the accelerated replication fork progression in Spi1/PU.1-overexpressing cells. These results identify a novel pathway by which an oncogene influences replication in the absence of DNA damage.

International audience; Chromosomal instability (CIN), a feature of most adult neoplasms from their early stages onward, is a driver of tumorigenesis. However, several malignancy subtypes, including some triple-negative breast cancers, display a paucity of genomic aberrations, thus suggesting that tumor development may occur in the absence of CIN. Here we show that the differentiation status of normal human mammary epithelial cells dictates cell behavior after an oncogenic event and predetermines the genetic routes toward malignancy. Whereas oncogene induction in differentiated cells induces massive DNA damage, mammary stem cells are resistant, owing to a preemptive program driven by the transcription factor ZEB1 and the methionine sulfoxide reductase MSRB3. The prevention of oncogene-induced DNA damage precludes induction of the oncosuppressive p53-dependent DNA-damage response, thereby increasing stem cells' intrinsic susceptibility to malignant transformation. In accord with this model, a subclass of breast neoplasms exhibit unique pathological features, including high ZEB1 expression, a low frequency of TP53 mutations and low CIN.

Xeroderma pigmentosum variant (XP-V) is a rare genetic disease, characterized by some sunlight sensitivity and predisposition to cutaneous malignancies. We described clinical and genetic features of the largest collection ever published of 23 XPV patients (ages between 21 and 86) from 20 unrelated families. Primary fibroblasts from patients showed normal nucleotide excision repair but UV-hypersensitivity in the presence of caffeine, a signature of the XP-V syndrome. 87% of patients developed skin tumors with a median age of 21 for the first occurrence. The median numbers of basal-cell carcinoma was 13 per patient, six for squamous-cell carcinoma, and five for melanoma. XP-V is due to defects in the translesion-synthesis DNA polymerase Polη coded by the POLH gene. DNA sequencing of POLH revealed 29 mutations, where 12 have not been previously identified, leading to truncated polymerases in 69% of patients. Four missense mutations are correlated with the protein stability by structural modeling of the Polη polymerase domain. There is a clear relationship between the types of missense mutations and clinical severity. For truncating mutations, which lead to an absence of or to inactive proteins, the life-cumulated UV exposure is probably the best predictor of cancer incidence, reinforcing the necessity to protect XP-Vs from sun exposure.

Human DNA polymerase iota (poliota) is a unique member of the Y-family of specialised polymerases that displays a 5'deoxyribose phosphate (dRP) lyase activity. Although poliota is well conserved in higher eukaryotes, its role in mammalian cells remains unclear. To investigate the biological importance of poliota in human cells, we generated fibroblasts stably downregulating poliota (MRC5-pol iota(KD)) and examined their response to several types of DNA-damaging agents. We show that cell lines downregulating poliota exhibit hypersensitivity to DNA damage induced by hydrogen peroxide (H(2)O(2)) or menadione but not to ethylmethane sulphonate (EMS), UVC or UVA. Interestingly, extracts from cells downregulating poliota show reduced base excision repair (BER) activity. In addition, poliota binds to chromatin after treatment of cells with H(2)O(2) and interacts with the BER factor XRCC1. Finally, green fluorescent protein-tagged poliota accumulates at the sites of oxidative DNA damage in living cells. This recruitment is partially mediated by its dRP lyase domain and ubiquitin-binding domains. These data reveal a novel role of human poliota in protecting cells from oxidative damage.

Translesion polymerase eta (polη) was characterized for its ability to replicate ultraviolet-induced DNA lesions that stall replicative polymerases, a process promoted by Rad18-dependent PCNA mono-ubiquitination. Recent findings have shown that polη also acts at intrinsically difficult to replicate sequences. However, the molecular mechanisms that regulate its access to these loci remain elusive. Here, we uncover that polη travels with replication forks during unchallenged S phase and this requires its SUMOylation on K163. Abrogation of polη SUMOylation results in replication defects in response to mild replication stress, leading to chromosome fragments in mitosis and damage transmission to daughter cells. Rad18 plays a pivotal role, independently of its ubiquitin ligase activity, acting as a molecular bridge between polη and the PIAS1 SUMO ligase to promote polη SUMOylation. Our results provide the first evidence that SUMOylation represents a new way to target polη to replication forks, independent of the Rad18-mediated PCNA ubiquitination, thereby preventing under-replicated DNA., Translesion synthesis polymerase eta has a well characterized role in replicating past UV-induced DNA lesions and has recently been shown to act at difficult to replicate sequences. Here the authors show that its SUMOylation is required to recruit pol eta at the replication fork and to prevent under-replicated DNA.

To study the relationships between different DNA repair pathways, we established a set of clones in which one specific DNA repair gene was silenced using long-term RNA interference in HeLa cell line. We focus here on genes involved in either nucleotide excision repair (XPA and XPC) or nonhomologous end joining (NHEJ; DNA-PKcs and XRCC4). As expected, XPAKD (knock down) and XPCKD cells were highly sensitive to UVC. DNA-PKcsKD and XRCC4KD cells presented an increased sensitivity to various inducers of double-strand breaks (DSBs) and a 70% to 80% reduction of in vitro NHEJ activity. Long-term silencing of XPC gene expression led to an increased sensitivity to etoposide, a topoisomerase II inhibitor that creates DSBs through the progression of DNA replication forks. XPCKD cells also showed intolerance toward acute γ-ray irradiation. We showed that XPCKD cells exhibited an altered spectrum of NHEJ products with decreased levels of intramolecular joined products. Moreover, in both XPCKD and DNA-PKcsKD cells, XRCC4 and ligase IV proteins were mobilized on damaged nuclear structures at lower doses of DSB inducer. In XPC-proficient cells, XPC protein was released from nuclear structures after induction of DSBs. By contrast, silencing of XPA gene expression did not have any effect on sensitivity to DSB or NHEJ. Our results suggest that XPC deficiency, certainly in combination with other genetic defects, may contribute to impair DSB repair. [Cancer Res 2007;67(6):2526–34]

Xeroderma pigmentosum variant (XP-V) is a rare genetic disease, characterized by sunlight sensitivity and predisposition to cutaneous malignancies. XP-V is caused by a deficiency in DNA polymerase eta (Polη) that plays a pivotal role in translesion synthesis by bypassing UV-induced pyrimidine dimers. Previously we identified a new Polη variant containing two missense mutations, one mutation within the bipartite NLS (T692A) and a second mutation on the stop codon (X714W) leading to a longer protein with an extra 8 amino acids (721 instead of 713 AA). First biochemical analysis revealed that this Polη missense variant was barely detectable by western blot. As this mutant is extremely unstable and is nearly undetectable, a definitive measure of its functional deficit in cells has not been explored. Here we report the molecular and cellular characterization of this missense variant. In cell free extracts, the extra 8 amino acids in the C-terminal of Polη(721) only slightly reduce the bypass efficiency through CPD lesions. In vivo, Polη(721) accumulates in replication factories and interacts with mUb-PCNA albeit at lower level than Polη(wt). XP-V cells overexpressing Polη(721) were only slightly UV-sensitive. Altogether, our data strongly suggest that Polη(721) is functional and that the patient displays a XP-V phenotype because the mutant protein is excessively unstable. We then investigated the molecular mechanisms involved in this excessive proteolysis. We showed that Polη(721) is degraded by the proteasome in an ubiquitin-dependent manner and that this proteolysis is independent of the E3 ligases, CRL4(cdt2) and Pirh2, reported to promote Polη degradation. We then demonstrated that the extra 8 amino acids of Polη(721) do not act as a degron but rather induce a conformational change of the Polη C-terminus exposing its bipartite NLS as well as a sequence close to its UBZ to the ubiquitin/proteasome system. Interestingly we showed that the clinically approved proteasome inhibitor, Bortezomib restores the levels of Polη(721) suggesting that this might be a therapeutic approach to preventing tumor development in certain XP-V patients harboring missense mutations.

The human KIN17 protein is a chromatin-associated protein involved in DNA replication. Certain tumor cell lines overproduce KIN17 protein. Among 16 cell lines, the highest KIN17 protein level was observed in H1299 non-small cell lung cancer cells, whereas the lowest was detected in MeWo melanoma cells. Cells displaying higher KIN17 protein levels exhibited elevated RPA70 protein contents. High KIN17 protein levels may be a consequence of the tumorigenic phenotype or a prerequisite for tumor progression. Twenty-four hours after exposure to ionizing radiation, after the completion of DNA repair, a co-induction of chromatin-bound KIN17 and RPA70 proteins was detected. Etoposide, an inhibitor of topoisomerase II generating double-strand breaks, triggered the concentration of KIN17 into punctuate intranuclear foci. KIN17 may be associated with unrepaired DNA sites. Flow cytometry analysis revealed that 48 h after transfection the uppermost KIN17-positive RKO cells shifted in the cell cycle toward higher DNA content, suggesting that KIN17 protein induced defects in chromatin conformation. Cells displaying reduced levels of KIN17 transcript exhibited a sixfold increased radiosensitivity at 2 Gy. The KIN17 protein may be a component of the DNA replication machinery that participates in the cellular response to unrepaired DSBs, and an impaired KIN17 pathway leads to an increased sensitivity to ionizing radiation.

Switching between replicative and translesion synthesis (TLS) DNA polymerases are crucial events for the completion of genomic DNA synthesis when the replication machinery encounters lesions in the DNA template. In eukaryotes, the translesional DNA polymerase η (Polη) plays a central role for accurate bypass of cyclobutane pyrimidine dimers, the predominant DNA lesions induced by ultraviolet irradiation. Polη deficiency is responsible for a variant form of the Xeroderma pigmentosum (XPV) syndrome, characterized by a predisposition to skin cancer. Here, we show that the FF483-484 amino acids in the human Polη (designated F1 motif) are necessary for the interaction of this TLS polymerase with POLD2, the B subunit of the replicative DNA polymerase δ, both in vitro and in vivo. Mutating this motif impairs Polη function in the bypass of both an N-2-acetylaminofluorene adduct and a TT-CPD lesion in cellular extracts. By complementing XPV cells with different forms of Polη, we show that the F1 motif contributes to the progression of DNA synthesis and to the cell survival after UV irradiation. We propose that the integrity of the F1 motif of Polη, necessary for the Polη/POLD2 interaction, is required for the establishment of an efficient TLS complex.

International audience; The SLX4 Fanconi anemia protein is a tumor suppres- sor that may act as a key regulator that engages the cell into specific genome maintenance pathways. Here, we show that the SLX4 complex is a SUMO E3 ligase that SUMOylates SLX4 itself and the XPF sub- unit of the DNA repair/recombination XPF-ERCC1 endonuclease. This SLX4-dependent activity is medi- ated by a remarkably specific interaction between SLX4 and the SUMO-charged E2 conjugating enzyme UBC9 and relies not only on newly identified SUMO- interacting motifs (SIMs) in SLX4 but also on its BTB domain. In contrast to its ubiquitin-binding UBZ4 motifs, SLX4 SIMs are dispensable for its DNA inter- strand crosslink repair functions. Instead, while detri- mental in response to global replication stress, the SUMO E3 ligase activity of the SLX4 complex is crit- ical to prevent mitotic catastrophe following common fragile site expression.

During translesion synthesis (TLS), low-fidelity polymerases of the Y-family polymerases bypass DNA damages that block the progression of conventional processive DNA polymerases, thereby allowing the completion of DNA replication. Among the TLS polymerases, DNA polymerase eta (polη) performs nucleotide incorporation past ultraviolet (UV) photoproducts and is deficient in cancer-prone xeroderma pigmentosum variant (XPV) syndrome. Upon UV irradiation, the DNA sliding clamp PCNA is monoubiquitylated on its conserved Lys-164. This event is considered to facilitate the TLS process in vivo since polη preferentially interacts with monoubiquitylated PCNA through its ubiquitin-binding domain (UBZ) as well as its PCNA interacting peptide (PIP)-box. However, recent observations questioned this model. Therefore, in this study, we re-examined the relative contribution of the regulatory UBZ and PIP domains of polη in response to UVC. We show that simultaneous invalidation of both motifs confers sensitivity to UVC, sensitization by low concentrations of caffeine, prolonged inhibition of DNA synthesis and persistent S phase checkpoint activation, all characteristic features of XPV cells. While each domain is essential for efficient accumulation of polη in replication factories, mutational inactivation of UBZ or PIP motif only confers a slight sensitivity to UVC indicating that, although informative, polη focus analysis is not a reliable tool to assess the polη's ability to function in TLS in vivo. Taken together, these data indicate that PIP and UBZ motifs are not required for recruitment but for retention of polη at sites of stalled replication forks. We propose that this is a way to ensure that a sufficient amount of the protein is available for its bypass function. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.

Posttranslational modification of PCNA by ubiquitin plays an important role in coordinating the processes of DNA damage tolerance during DNA replication. The monoubiquitination of PCNA was shown to facilitate the switch between the replicative DNA polymerase with the low-fidelity polymerase eta (η) to bypass UV-induced DNA lesions during replication. Here, we show that in response to oxidative stress, PCNA becomes transiently monoubiquitinated in an S phase- and USP1-independent manner. Moreover, Polη interacts with mUb-PCNA at sites of oxidative DNA damage via its PCNA-binding and ubiquitin-binding motifs. Strikingly, while functional base excision repair is not required for this modification of PCNA or Polη recruitment to chromatin, the presence of hMsh2-hMsh6 is indispensable. Our findings highlight an alternative pathway in response to oxidative DNA damage that may coordinate the removal of oxidatively induced clustered DNA lesions and could explain the high levels of oxidized DNA lesions in MSH2-deficient cells.

DNA polymerase eta (poleta) performs translesion synthesis past ultraviolet (UV) photoproducts and is deficient in cancer-prone xeroderma pigmentosum variant (XP-V) syndrome. The slight sensitivity of XP-V cells to UV is dramatically enhanced by low concentrations of caffeine. So far, the biological explanation for this feature remains elusive. Using DNA combing, we showed that translesion synthesis defect leads to a strong reduction in the number of active replication forks and a high proportion of stalled forks in human cells, which contrasts with budding yeast. Moreover, extensive regions of single-strand DNA are formed during replication in irradiated XP-V cells, leading to an over-activation of ATR/Chk1 pathway after low UVC doses. Addition of a low concentration of caffeine post-irradiation, although inefficient to restore S-phase progression, significantly decreases Chk1 activation and abrogates DNA synthesis in XP-V cells. While inhibition of Chk1 activity by UCN-01 prevents UVC-induced S-phase delay in wild-type cells, it aggravates replication defect in XP-V cells by increasing fork stalling. Consequently, UCN-01 sensitizes XP-V cells to UVC as caffeine does. Our findings indicate that poleta acts at stalled forks to resume their progression, preventing the requirement for efficient replication checkpoint after low UVC doses. In the absence of poleta, Chk1 kinase becomes essential for replication resumption by alternative pathways, via fork stabilization.

We developed and characterized replicative small interfering RNA (siRNA) vectors for efficient, specific, and long-term gene silencing in human cells. We created stable XPAKD and XPCKD (knockdown) syngeneic cell lines to mimic human cancer-prone syndromes. We also silenced HSAKIN17. Several clones displaying undetectable protein levels of XPA, XPC, or HSAkin17 were grown for more than 300 days. This stability of gene silencing over several months of culture allows us to assess the specific involvement of these proteins in UVC sensitivity in syngeneic cells. Unlike XPA, HSAKIN17, and XPC gene silencing dramatically impeded HeLa cell growth for several weeks after transfection. As expected, XPAKD and XPCKD HeLa cells were highly UVC sensitive. They presented an impaired unscheduled DNA synthesis after UVC irradiation. Interestingly, XPCKD HeLa clones were more sensitive to UVC than their XPAKD or KIN17KD counterparts. Hygromycin B withdrawal led to the total disappearance of EBV vectors and the resumption of normal XPA or XPC protein levels. Whereas reverted XPAKD cells recovered a normal UVC sensitivity, XPCKD cells remained highly sensitive, suggestive of irreversible damage following long-term XPC silencing. Our results show that in HeLa cells, HSAkin17 participates indirectly in early events following UVC irradiation, and XPC deficiency strongly affects cell physiology and contributes to UVC sensitivity to a greater extent than does XPA. EBV-based siRNA vectors improve the interest of siRNA by permitting long-term gene silencing without the safety concerns inherent in viral-based siRNA vehicles.

The Homo sapiens kin17 ((HSA)kin17) protein is a chromatin-associated protein conserved during evolution and overproduced in certain human tumor cell lines. For the first time, immunoelectron microscopy analysis of endogenous (HSA)kin17 protein revealed an ultrastructural co-localization of (HSA)kin17 and bromodeoxyuridine (BrdUrd) at sites of DNA replication after either short (15 min) or long (120 min) pulses of BrdUrd labeling. After hydroxyurea (HU) or L-mimosine (Mimo) block and withdrawal, we observed that (HSA)kin17 was recruited onto the chromatin during the re-entry and the progression in the S phase. These results are consistent with a major role of (HSA)kin17 protein in DNA replication factories. Other treatments hampering replication fork progression and/or inducing double-strand breaks also triggered an accumulation and a concentration of the chromatin-bound (HSA)kin17 protein into large intranuclear foci 24 h post-treatment. Moreover, HU- and Mimo-induced (HSA)kin17 foci were retained in the nucleus after detergent extraction, suggesting a strong association with nuclear structures. Gel filtration analyses of cellular extracts showed that endogenous (HSA)kin17 protein co-eluted with both replication proteins RPA32 and RPA70 in a fraction containing complexes of M(r) 600,000. Interestingly, HU-induced G(1)-S arrest triggered an increase in the molecular weight of complexes containing (HSA)kin17 protein. Hence, treatments interfering with either initiation and/or elongation of DNA replication also recruited chromatin-bound (HSA)kin17 protein. We hypothesize that in the presence of unrepaired DNA damage, (HSA)kin17 protein concentrated into high molecular weight complexes probably to create a bridge that contributes to the harmonization of DNA replication and repair.

The human DNA-binding (HSA)kin17 protein cross-reacts with antibodies raised against the stress-activated Escherichia coli RecA protein. We show here that (HSA)kin17 protein is directly associated with chromosomal DNA as judged by cross-linking experiments on living cells. We detected increased amounts of DNA-bound (HSA)kin17 protein 24 h after gamma irradiation, with 2.6-fold more (HSA)kin17 molecules after 6 Gy of irradiation (46,000-117,000 molecules). At this time we observed that highly proliferating RKO cells displayed the concentration and co-localization of (HSA)kin17 and replication protein A in nucleoplasmic foci. Our results suggest that 24 h post-irradiation (HSA)kin17 protein may localize at the sites of unrepaired DNA damages. RKO clones expressing an (HSA)KIN17 antisense transcript (RASK.5 and RASK.13 cells) revealed that reduced (HSA)kin17 protein levels are correlated with a decrease in clonogenic cell growth and cell proliferation, as well as an accumulation of cells in early and mid-S phase. Taken together our observations support the idea that (HSA)kin17 protein is a DNA maintenance protein involved in the cellular response to the presence of DNA damage and suggest that it helps to overcome the perturbation of DNA replication produced by unrepaired lesions.

Complete and accurate DNA replication is crucial to transfer correct genetic information to the daughter cells. Various obstacles can interfere with the progression of the replication machinery, threatening genome integrity. Specialized error-prone translesion DNA polymerases (TLS polymerases) assist the replicative polymerases to replicate across DNA lesions. During my PhD I characterized the contribution of TLS pol eta (polη), best known for its role in preventing UV-induced mutagenesis, during unperturbed replication. Polη was shown to promote the stability of the common fragile sites and associates with the replisome in unchallenged S phase. However, the kind of replication barriers requiring pol eta and the consequences of its absence on the replication of these regions were unclear. My results show that polη is recruited at a subset of replication forks all along the S phase and that polη defect modifies the replication timing of genomic regions enriched in large transcribed genes, where transcription-replication conflicts (TRCs) are more likely to occur. Overall, I show that polη recruitment at the replication fork is transcription-dependent, and that pol eta plays a role in the coping with TRCs. Altogether, these results highlight a new role for an error-prone DNA polymerase in protecting the genome stability., La réplication complète et fidèle de l’ADN est cruciale pour transmettre l’information génétique de manière correcte aux cellules filles. Divers obstacles peuvent interférer avec la progression de la machinerie de réplication, et donc menacer l’intégrité du génome. Des ADN polymérases spécialisées, dites translésionnelles (polymérases TLS), assistent les ADN polymérases réplicatives pour la poursuite de la réplication malgré ces lésions. Elles peuvent répliquer de manière fidèle ou non ces entraves, mais sont mutagènes sur des séquences d’ADN non-endommagées. Au cours de ma thèse, j’ai pu caractériser davantage la contribution de l’ADN polymérase TLS eta (polη) au cours de la réplication non-perturbée. Cette polymérase permet principalement de prévenir la mutagénèse induite par les UV. Mais il a également été montré qu’elle promeut la stabilité des sites fragiles communs, et est associée au réplisome durant la phase S non-perturbée. Cependant, la nature des obstacles nécessitant polη et les conséquences de son absence pour la réplication de ces régions restaient à déterminer. Mes résultats montrent que polη est recrutée au niveau d’une fraction des fourches de réplication tout au long de la phase S et que l’absence de pol eta conduit à une modification du timing de réplication de régions génomiques riches en grands gènes transcrits, où les conflits entre réplication et transcription sont potentiellement plus fréquents. Plus généralement, je montre que le recrutement de pol eta à la fourche de réplication dépend de la transcription et qu’elle joue un rôle dans la prise en charge des conflits entre réplication et transcription. Ces résultats mettent en évidence un nouveau rôle de protection de la stabilité du génome pour cette ADN polymérase mutagène.