DNA damage induced Pol η recruitment takes place independently of the cell cycle phase

When DNA is damaged in cells progressing through S phase, replication blockage can be avoided by TLS (Translesion DNA synthesis). This is an auxiliary replication mechanism that relies on the function of specialized polymerases that accomplish DNA damage bypass. Intriguingly, recent evidence has linked TLS polymerases to processes that can also take place outside S phase such as nucleotide excision repair (NER). Here we show that Pol η is recruited to UV-induced DNA lesions in cells outside S phase including cells permanently arrested in G1. This observation was confirmed by different strategies including global UV irradiation, local UV irradiation and local multi-photon laser irradiation of single nuclei in living cells. The potential connection between Pol η recruitment to DNA lesions outside S phase and NER was further evaluated. Interestingly, the recruitment of Pol η to damage sites outside S phase did not depend on active NER, as UV-induced focus formation occurred normally in XPA, XPG and XPF deficient fibroblasts. Our data reveals that the re-localization of the TLS polymerase Pol η to photo-lesions might be temporally and mechanistically uncoupled from replicative DNA synthesis and from DNA damage processing.     

Cooperation of RAD51 and RAD54 in regression of a model replication fork

DNA lesions cause stalling of DNA replication forks, which can be lethal for the cell. Homologous recombination (HR) plays an important role in DNA lesion bypass. It is thought that Rad51, a key protein of HR, contributes to the DNA lesion bypass through its DNA strand invasion activity. Here, using model stalled replication forks we found that RAD51 and RAD54 by acting together can promote DNA lesion bypass in vitro through the 'template-strand switch' mechanism. This mechanism involves replication fork regression into a Holliday junction ('chicken foot structure'), DNA synthesis using the nascent lagging DNA strand as a template and fork restoration. Our results demonstrate that RAD54 can catalyze both regression and restoration of model replication forks through its branch migration activity, but shows strong bias toward fork restoration. We find that RAD51 modulates this reaction; by inhibiting fork restoration and stimulating fork regression it promotes accumulation of the chicken foot structure, which we show is essential for DNA lesion bypass by DNA polymerase in vitro. These results indicate that RAD51 in cooperation with RAD54 may have a new role in DNA lesion bypass that is distinct from DNA strand invasion.     

Changes in the architecture and abundance of replication intermediates delineate the chronology of DNA damage tolerance pathways at UV-stalled replication forks in human cells

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.     

The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis

Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.     

Recombination at Mammalian Telomeres: An Alternative Mechanism for Telomere Protection and Elongation

In mammalian cells, homologous recombination (HR) provides anaccurate mechanism for the repair of DNA double-strand breaks causedby replication fork breakdown or DNA damaging agents. HR also plays arole in the maintenance of eukaryotic telomeres; cells defective in therecombinational repair proteins RAD51D or RAD54 exhibit telomereshortening and end-to-end chromosome fusions. Here we discuss theway in which HR contributes to telomere protection and elongation inmammalian cells. Understanding the mechanisms by which HRpromotes telomere maintenance has important implications for genomicstability and tumorigenesis.     

Gap-filling and bypass at the replication fork are both active mechanisms for tolerance of low-dose ultraviolet-induced DNA damage in the human genome

Ultraviolet (UV)-induced DNA damage are removed by nucleotide excision repair (NER) or can be tolerated by specialized translesion synthesis (TLS) polymerases, such as Polη. TLS may act at stalled replication forks or through an S-phase independent gap-filling mechanism. After UVC irradiation, Polη-deficient (XP-V) human cells were arrested in early S-phase and exhibited both single-strand DNA (ssDNA) and prolonged replication fork stalling, as detected by DNA fiber assay. In contrast, NER deficiency in XP-C cells caused no apparent defect in S-phase progression despite the accumulation of ssDNA and a G2-phase arrest. These data indicate that while Polη is essential for DNA synthesis at ongoing damaged replication forks, NER deficiency might unmask the involvement of tolerance pathway through a gap-filling mechanism. ATR knock down by siRNA or caffeine addition provoked increased cell death in both XP-V and XP-C cells exposed to low-dose of UVC, underscoring the involvement of ATR/Chk1 pathway in both DNA damage tolerance mechanisms. We generated a unique human cell line deficient in XPC and Polη proteins, which exhibited both S- and G2-phase arrest after UVC irradiation, consistent with both single deficiencies. In these XP-C/Polη^KD cells, UVC-induced replicative intermediates may collapse into double-strand breaks, leading to cell death. In conclusion, both TLS at stalled replication forks and gap-filling are active mechanisms for the tolerance of UVC-induced DNA damage in human cells and the preference for one or another pathway depends on the cellular genotype.     

RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells

Translesion DNA synthesis (TLS) and homologous DNA recombination (HR) are two major pathways that account for survival after post-replicational DNA damage. TLS functions by filling gaps on a daughter strand that remain after DNA replication caused by damage on the mother strand, while HR can repair gaps and breaks using the intact sister chromatid as a template. The RAD18 gene, which is conserved from lower eukaryotes to vertebrates, is essential for TLS in Saccharomyces cerevisiae. To investigate the role of RAD18, we disrupted RAD18 by gene targeting in the chicken B-lymphocyte line DT40. RAD18(-/-) cells are sensitive to various DNA-damaging agents including ultraviolet light and the cross-linking agent cisplatin, consistent with its role in TLS. Interestingly, elevated sister chromatid exchange, which reflects HR- mediated post-replicational repair, was observed in RAD18(-/-) cells during the cell cycle. Strikingly, double mutants of RAD18 and RAD54, a gene involved in HR, are synthetic lethal, although the single mutant in either gene can proliferate with nearly normal kinetics. These data suggest that RAD18 plays an essential role in maintaining chromosomal DNA in cooperation with the RAD54-dependent DNA repair pathway.     

The roles of PCNA SUMOylation, Mms2-Ubc13 and Rad5 in translesion DNA synthesis in Saccharomyces cerevisiae

Mms2, in concert with Ubc13 and Rad5, is responsible for polyubiquitination of replication processivity factor PCNA. This modification activates recombination-like DNA damage-avoidance mechanisms, which function in an error-free manner. Cells deprived of Mms2, Ubc13 or Rad5 exhibit mutator phenotypes as a result of the channelling of premutational DNA lesions to often error-prone translesion DNA synthesis (TLS). Here we show that Siz1-mediated PCNA SUMOylation is required for the stimulation of this TLS, despite the presence of PCNA monoubiquitination. The stimulation of spontaneous mutagenesis by Siz1 in cells carrying rad5 and/or mms2 mutations is connected with the known role of PCNA SUMOylation in the inhibition of Rad52-mediated recombination. However, following UV irradiation, Siz1 is engaged in additional, as yet undefined, mechanisms controlling genetic stability at the replication fork. We also demonstrate that in the absence of PCNA SUMOylation, Mms2-Ubc13 and Rad5 may, independently of each other, function in the stimulation of TLS. Based on this finding and on an analysis of the epistatic relationships between SIZ1, MMS2 and RAD5, with respect to UV sensitivity, we conclude that PCNA SUMOylation is responsible for the functional differences between the Mms2 and Rad5 homologues of Saccharomyces cerevisiae and Schizosaccharomyces pombe.     

RAD51 is involved in repair of damage associated with DNA replication in mammalian cells

The RAD51 protein, a eukaryotic homologue of the Escherichia coli RecA protein, plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in mammalian cells. Recent findings suggest that HR may be important in repair following replication arrest in mammalian cells. Here, we have investigated the role of RAD51 in the repair of different types of damage induced during DNA replication with etoposide, hydroxyurea or thymidine. We show that etoposide induces DSBs at newly replicated DNA more frequently than gamma-rays, and that these DSBs are different from those induced by hydroxyurea. No DSB was found following treatment with thymidine. Although these compounds appear to induce different DNA lesions during DNA replication, we show that a cell line overexpressing RAD51 is resistant to all of them, indicating that RAD51 is involved in repair of a wide range of DNA lesions during DNA replication. We observe fewer etoposide-induced DSBs in RAD51-overexpressing cells and that HR repair of etoposide-induced DSBs is faster. Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although global HR appears not to be suppressed. This suggests that overexpression of RAD51 prevents long-tract HR occurring during DNA replication. We discuss our results in light of recent models suggested for HR at stalled replication forks.     

Polymerase zeta dependency of increased adaptive mutation frequencies in nucleotide excision repair-deficient yeast strains

Reversions of an auxotrophy-causing frameshift allele during prolonged starvation of yeast cells were used as a means to elucidate the mechanisms concerned with the generation of spontaneous adaptive mutations in cell cycle-arrested cells. Whereas about 50% of these reversions were previously shown to depend on the non-homologous end joining pathway of DNA double-strand break repair, the origin of the residual 50% remains unknown. In search for a mechanism for generation of the latter fraction of reversions we examined the role of the translesion synthesis (TLS) polymerases zeta, eta and Rev1p in cells with wild-type or impaired nucleotide excision repair (NER) capacity. The basal level of adaptive mutations in the repair-proficient wild type was not influenced by disruptions of the genes coding for these three TLS polymerases. Intriguingly, a deficiency in NER by disruption of RAD14, RAD16 or RAD26 resulted in a significantly higher frequency of adaptive mutation, yet this increase was strictly dependent on an intact REV3 gene, coding for the catalytic subunit of polymerase zeta. Furthermore, we observed that intact REV3 was also required for the occurrence of increased frequencies of adaptive mutants in the NER-proficient wild type following UV irradiation. While in proliferating cells the translesion synthesis function of polymerase zeta is connected to DNA replication, our data suggest that in cell cycle-arrested cells this enzyme is able to carry out either TLS or error-prone polymerization along an undamaged template in the course of repair processes. Such a hitherto unappreciated activity of polymerase zeta in non-replicating cells may contribute to the incidence of mutations in evolution, aging and cancer.     

Human DNA polymerase eta promotes DNA synthesis from strand invasion intermediates of homologous recombination

Stalled replication forks pose a serious threat to genome integrity. To overcome the catastrophic consequences associated with fork demise, translesion synthesis (TLS) polymerases such as poleta promote DNA synthesis past lesions. Alternatively, a stalled fork may collapse and undergo repair by homologous recombination. By using fractionated cell extracts and purified recombinant proteins, we show that poleta extends DNA synthesis from D loop recombination intermediates in which an invading strand serves as the primer. Extracts from XP-V cells, which are defective in poleta, exhibit severely reduced D loop extension activity. The D loop extension activity of poleta is unusual, as this reaction cannot be promoted by the replicative DNA polymerase delta or by other TLS polymerases such as poliota. Moreover, we find that poleta interacts with RAD51 recombinase and RAD51 stimulates poleta-mediated D loop extension. Our results indicate a dual function for poleta at stalled replication forks: the promotion of translesion synthesis and the reinitiation of DNA synthesis by homologous recombination repair.     

Building up and breaking down: mechanisms controlling recombination during replication

The complete and faithful duplication of the genome is an essential prerequisite for proliferating cells to maintain genome integrity. This objective is greatly challenged by DNA damage encountered during replication, which causes fork stalling and in certain cases, fork breakage. DNA damage tolerance (DDT) pathways mitigate the effects on fork stability induced by replication fork stalling by mediating damage-bypass and replication fork restart. These DDT mechanisms, largely relying on homologous recombination (HR) and specialized polymerases, can however contribute to genome rearrangements and mutagenesis. There is a profound connection between replication and recombination: recombination proteins protect replication forks from nuclease-mediated degradation of the nascent DNA strands and facilitate replication completion in cells challenged by DNA damage. Moreover, in case of fork collapse and formation of double strand breaks (DSBs), the recombination factors present or recruited to the fork facilitate HR-mediated DSB repair, which is primarily error-free. Disruption of HR is inexorably linked to genome instability, but the premature activation of HR during replication often leads to genome rearrangements. Faithful replication necessitates the downregulation of HR and disruption of active RAD51 filaments at replication forks, but upon persistent fork stalling, building up of HR is critical for the reorganization of the replication fork and for filling-in of the gaps associated with discontinuous replication induced by DNA lesions. Here we summarize and reflect on our understanding of the mechanisms that either suppress recombination or locally enhance it during replication, and the principles that underlie this regulation.     

Mismatch repair protein MSH2 regulates translesion DNA synthesis following exposure of cells to UV radiation

Translesion DNA synthesis (TLS) can use specialized DNA polymerases to insert and/or extend nucleotides across lesions, thereby limiting stalled replication fork collapse and the potential for cell death. Recent studies have shown that monoubiquitinated proliferating cell nuclear antigen (PCNA) plays an important role in recruitment of Y-family TLS polymerases to stalled replication forks after DNA damage treatment. To explore the possible roles of other factors that regulate the ultraviolet (UV)-induced assembly of specialized DNA polymerases at arrested replication forks, we performed immunoprecipitation experiments combined with mass spectrometry and established that DNA polymerase kappa (Polκ) can partner with MSH2, an important mismatch repair protein associated with hereditary non-polyposis colorectal cancer. We found that depletion of MSH2 impairs PCNA monoubiquitination and the formation of foci containing Polκ and other TLS polymerases after UV irradiation of cells. Interestingly, expression of MSH2 in Rad18-deficient cells increased UV-induced Polκ and REV1 focus formation without detectable changes in PCNA monoubiquitination, indicating that MSH2 can regulate post-UV focus formation by specialized DNA polymerases in both PCNA monoubiquitination-dependent and -independent fashions. Moreover, we observed that MSH2 can facilitate TLS across cyclobutane pyrimidine dimers photoproducts in living cells, presenting a novel role of MSH2 in post-UV cellular responses.     

Non‐recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance

DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error‐prone, HR uses the sister chromatid and is mostly error‐free. We report that the HR protein Rad52—but not Rad51 and Rad57—acts in concert with the TLS machinery (Rad6/Rad18‐mediated PCNA ubiquitylation and polymerases Rev1/Pol ζ) to repair MMS and UV light‐induced ssDNA gaps through a non‐recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS‐ and UV‐induced HR); accordingly, Rad52 is required for efficient DNA damage‐induced mutagenesis. In addition, Rad52, Rad51, and Rad57, but not Rad54, facilitate Rad6/Rad18 binding to chromatin and subsequent DNA damage‐induced PCNA ubiquitylation. Therefore, Rad52 facilitates the tolerance process not only by HR but also by TLS through Rad51/Rad57‐dependent and ‐independent processes, providing a novel role for the recombination proteins in maintaining genome integrity.     

Interaction of human DNA polymerase eta with monoubiquitinated PCNA: a possible mechanism for the polymerase switch in response to DNA damage

Most types of DNA damage block replication fork progression during DNA synthesis because replicative DNA polymerases are unable to accommodate altered DNA bases in their active sites. To overcome this block, eukaryotic cells employ specialized translesion synthesis (TLS) polymerases, which can insert nucleotides opposite damaged bases. In particular, TLS by DNA polymerase eta (poleta) is the major pathway for bypassing UV photoproducts. How the cell switches from replicative to TLS polymerase at the site of blocked forks is unknown. We show that, in human cells, PCNA becomes monoubiquitinated following UV irradiation of the cells and that this is dependent on the hRad18 protein. Monoubiquitinated PCNA but not unmodified PCNA specifically interacts with poleta, and we have identified two motifs in poleta that are involved in this interaction. Our findings provide an attractive mechanism by which monoubiquitination of PCNA might mediate the polymerase switch.     

Multiple roles of Rev3, the catalytic subunit of polzeta in maintaining genome stability in vertebrates

Translesion DNA synthesis (TLS) and homologous DNA recombination (HR) are two major postreplicational repair (PRR) pathways. The REV3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase zeta, which is involved in mutagenic TLS. To investigate the role of REV3 in vertebrates, we disruped the gene in chicken DT40 cells. REV3(-/-) cells are sensitive to various DNA-damaging agents, including UV, methyl methanesulphonate (MMS), cisplatin and ionizing radiation (IR), consistent with its role in TLS. Interestingly, REV3(-/-) cells showed reduced gene targeting efficiencies and significant increase in the level of chromosomal breaks in the subsequent M phase after IR in the G(2) phase, suggesting the involvement of Rev3 in HR-mediated double-strand break repair. REV3(-/-) cells showed significant increase in sister chromatid exchange events and chromosomal breaks even in the absence of exogenous genotoxic stress. Furthermore, double mutants of REV3 and RAD54, genes involved in HR, are synthetic lethal. In conclusion, Rev3 plays critical roles in PRR, which accounts for survival on naturally occurring endogenous as well as induced damages during replication.