Pol η–dependent DNA synthesis at stalled replication forks during S phase suppresses chronic fragile site instability by preventing checkpoint-blind under-replicated DNA in mitosis.

"Replicative stress" is one of the main factors underlying neoplasia from its early stages. Genes involved in DNA synthesis may therefore represent an underexplored source of potential prognostic markers for cancer. To this aim, we generated gene expression profiles from two independent cohorts (France, n = 206; United Kingdom, n = 117) of patients with previously untreated primary breast cancers. We report here that among the 13 human nuclear DNA polymerase genes, DNA Polymerase θ (POLQ) is the only one significantly up-regulated in breast cancer compared with normal breast tissues. Importantly, POLQ up-regulation significantly correlates with poor clinical outcome (4.3-fold increased risk of death in patients with high POLQ expression), and this correlation is independent of Cyclin E expression or the number of positive nodes, which are currently considered as markers for poor outcome. POLQ expression provides thus an additional indicator for the survival outcome of patients with high Cyclin E tumor expression or high number of positive lymph nodes. Furthermore, to decipher the molecular consequences of POLQ up-regulation in breast cancer, we generated human MRC5-SV cell lines that stably overexpress POLQ. Strong POLQ expression was directly associated with defective DNA replication fork progression and chromosomal damage. Therefore, POLQ overexpression may be a promising genetic instability and prognostic marker for breast cancer.specialized DNA replication | prognosis marker | S-phase checkpoint B esides the "replicative" DNA polymerases POLA, POLD, and POLE, which are involved in conventional DNA replication of the undamaged genome, mammalian nuclei contain 10 additional specialized DNA polymerases that play a role in replication, repair, and recombination of damaged DNA (1, 2) and thus may be of paramount importance to preserve the integrity of the genome.Specialized DNA polymerases are frequently deregulated in neoplasia (3-10). Indeed, the intracellular balance between the error-free, replicative polymerases POLA, POLD, and POLE and the error-prone, specialized DNA polymerases (POLH, POLL, POLM, POLN, POLK, POLB, POLI, POLQ, POLZ/REV3L, and REV1) appears to be of great importance for the maintenance of genome stability (11)(12)(13)(14). Here, we wondered whether misregulation of DNA polymerases could be a signature of breast cancer progression. Indeed, beside the standard classification used by pathologists, there is a clear lack of tools to accurately predict the clinical outcome of many patients.We specifically measured the expression levels of the 13 human replicative and specialized DNA polymerases in 206 breast carcinomas. We report that, differently from the replicative and the other specialized DNA polymerases, POLQ was significantly upregulated in most of the breast tumors analyzed. Such up-regulation was associated with poor clinical outcome.POLQ is an error-prone, specialized DNA polymerase that might operate during "normal" genomic replication because it bypasses some endogenous DNA lesions an...

DNA polymerase ␤ (pol ␤) is the most error prone of all known eukaryotic DNA polymerases tested in vitro. Here, we show that cells overexpressing pol ␤ 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 ␤. 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 ␤ overexpression in cancer predisposition and tumor progression during chemotherapy.

Oncogene-induced replication stress (RS) promotes cancer development but also impedes tumor growth by activating anti-cancer barriers. To determine how cancer cells adapt to RS, we have monitored the expression of different components of the ATR-CHK1 pathway in primary tumor samples. We show that unlike upstream components of the pathway, the checkpoint mediators Claspin and Timeless are overexpressed in a coordinated manner. Remarkably, reducing the levels of Claspin and Timeless in HCT116 cells to pretumoral levels impeded fork progression without affecting checkpoint signaling. These data indicate that high level of Claspin and Timeless increase RS tolerance by protecting replication forks in cancer cells. Moreover, we report that primary fibroblasts adapt to oncogene-induced RS by spontaneously overexpressing Claspin and Timeless, independently of ATR signaling. Altogether, these data indicate that enhanced levels of Claspin and Timeless represent a gain of function that protects cancer cells from of oncogene-induced RS in a checkpoint-independent manner.

Accurate DNA replication during S-phase is fundamental to maintain genome integrity. During this critical process, replication forks frequently encounter obstacles that impede their progression. While the regulatory pathways which act in response to exogenous replication stress are beginning to emerge, the mechanisms by which fork integrity is maintained at naturally occurring endogenous replication-impeding sequences remains obscure. Notably, little is known about how cells replicate through special chromosomal regions containing structured non-B DNA, e.g. G4 quartets, known to hamper fork progression or trigger chromosomal rearrangements. Here, we have investigated the role in this process of the human translesion synthesis (TLS) DNA polymerases of the Y-family (pol η, pol ι, and pol κ), specialized enzymes known to synthesize DNA through DNA damage. We show that depletion by RNA interference of expression of the genes for Pol η or Pol κ, but not Pol ι, sensitizes U2OS cells treated with the G4-tetraplex interactive compound telomestatin and triggers double-strand breaks in HeLa cells harbouring multiple copies of a G-rich sequence from the promoter region of the human c-MYC gene, chromosomally integrated as a transgene. Moreover, we found that downregulation of Pol κ only raises the level of DSB in HeLa cells containing either one of two breakage hotspot structured DNA sequences in the chromosome, the major break region (Mbr) of BCL-2 gene and the GA rich region from the far right-hand end of the genome of the Kaposi Sarcoma associated Herpesvirus. These data suggest that naturally occurring DNA structures are physiological substrates of both pol η and pol κ. We discuss these data in the light of their downregulation in human cancers.

We have examined the capacity of calf thymus DNA polymerases a, f, 6, and e to perform in vitro translesion synthesis on a substrate containing a single d(GpG)-cisplatin adduct placed on codon 13 of the human HRAS gene. We found that DNA synthesis catalyzed by DNA polymerases ca, 6, and E was blocked at the base preceding the lesion Among the possible mechanisms of mutagenesis is error-prone replication by cellular DNA polymerases past a DNA lesion followed by fixation of the mutation during subsequent rounds of replication. In eukaryotic cells, it is still unclear whether the replicative DNA polymerases a, 8, and E can carry out translesion synthesis either alone or with the help of accessory proteins. Alternatively, a separate DNA polymerase may be required for this process. For example, genetic evidence in the yeast Saccharomyces cerevisiae indicates that a putative DNA polymerase, the product of the REV3 gene, may be involved in error-prone translesion synthesis but not in normal DNA replication (1). DNA polymerase (3 is one of the five mammalian polymerases identified to date and is believed to function primarily in the repair of damaged DNA (2). However, DNA polymerase a may also have a role in replicative synthesis, since the enzyme can substitute for DNA polymerase I during DNA replication in Escherichia coli (3), and it is required for the conversion of single-stranded M13 DNA to double-stranded DNA in Xenopus oocytes and in oocyte nuclear extracts (4). cis-Diamminedichloroplatinum(II) (cisplatin) is an anticancer agent widely used in the treatment of ovarian, testicular, head, and neck carcinomas (5). It is believed that this compound exerts its cytotoxic properties by forming stable lesions on DNA, primarily intrastrand cross-links at the N-7 positions of adjacent guanine bases [d(GpG)-cisplatin or Pt-d(GpG)] (6). Replicative bypass of cisplatin adducts has been described in bacteria (7,8) and in eukaryotic cells (9). Recent work in our laboratory has demonstrated that a single-stranded DNA The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.vector bearing a unique intrastrand bifunctional adduct Ptd(GpG) at codon 13 of the human protooncogene HRAS is replicated in simian COS-7 cells and that such translesion synthesis may be mutagenic (10).To our knowledge, the capacities of the major mammalian replication enzymes to bypass the Pt-d(GpG) lesion have not been compared. To address this question, we have investigated the ability of purified calf thymus DNA polymerases a, (3, 8, and s to catalyze in vitro the bypass synthesis of a single Pt-d(GpG) adduct placed on codon 13 of the human HRAS protooncogene, the same sequence used for our previous in vivo studies (10). Results show that only DNA polymerase 3 is capable of in vitro translesional synthesis and indicate that its ability to initiate DNA replication opposite the cisplatin adduct may ...

Failure to restart replication forks stalled at genomic regions that are difficult to replicate or contain endogenous DNA lesions is a hallmark of BRCA2 deficiency. The nucleolytic activity of MUS81 endonuclease is required for replication fork restart under replication stress elicited by exogenous treatments. Here we investigate whether MUS81 could similarly facilitate DNA replication in the context of BRCA2 abrogation. Our results demonstrate that replication fork progression in BRCA2-deficient cells requires MUS81. Failure to complete genome replication and defective checkpoint surveillance enables BRCA2-deficient cells to progress through mitosis with under-replicated DNA, which elicits severe chromosome interlinking in anaphase. MUS81 nucleolytic activity is required to activate compensatory DNA synthesis during mitosis and to resolve mitotic interlinks, thus facilitating chromosome segregation. We propose that MUS81 provides a mechanism of replication stress tolerance, which sustains survival of BRCA2-deficient cells and can be exploited therapeutically through development of specific inhibitors of MUS81 nuclease activity.

SUMMARY Cancer cells rely on the activation of telomerase or the alternative lengthening of telomeres (ALT) pathways for telomere maintenance and survival. ALT involves homologous recombination (HR)-dependent exchange and/or HR-associated synthesis of telomeric DNA. Utilizing proximity-dependent biotinylation (BioID), we sought to determine the proteome of telomeres in cancer cells that employ these distinct telomere elongation mechanisms. Our analysis reveals that multiple DNA repair networks converge at ALT telomeres. These include the specialized translesion DNA synthesis (TLS) proteins FANCJ-RAD18-PCNA and, most notably, DNA polymerase eta (Polη). We observe that the depletion of Polη leads to increased ALT activity and late DNA polymerase δ (Polδ)-dependent synthesis of telomeric DNA in mitosis. We propose that Polη fulfills an important role in managing replicative stress at ALT telomeres, maintaining telomere recombination at tolerable levels and stimulating DNA synthesis by Polδ.