Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing.

The effectiveness of checkpoint kinase 1 (Chk1) inhibitors at killing cancer cells is considered to be fully dependent on their effect on DNA replication initiation. Chk1 inhibition boosts origin firing, presumably limiting the availability of nucleotides and in turn provoking the slowdown and subsequent collapse of forks, thus decreasing cell viability. Here we show that slow fork progression in Chk1-inhibited cells is not an indirect effect of excess new origin firing. Instead, fork slowdown results from the accumulation of replication barriers, whose bypass is impeded by CDK-dependent phosphorylation of the specialized DNA polymerase eta (Polη). Also in contrast to the linear model, the accumulation of DNA damage in Chk1-deficient cells depends on origin density but is largely independent of fork speed. Notwithstanding this, origin dysregulation contributes only mildly to the poor proliferation rates of Chk1-depleted cells. Moreover, elimination of replication barriers by downregulation of helicase components, but not their bypass by Polη, improves cell survival. Our results thus shed light on the molecular basis of the sensitivity of tumors to Chk1 inhibition.

DNA polymerase κ-dependent DNA synthesis at stalled replication forks is important for CHK1 activation.

Formation of primed single-stranded DNA at stalled replication forks triggers activation of the replication checkpoint signalling cascade resulting in the ATR-mediated phosphorylation of the Chk1 protein kinase, thus preventing genomic instability. By using siRNA-mediated depletion in human cells and immunodepletion and reconstitution experiments in Xenopus egg extracts, we report that the Y-family translesion (TLS) DNA polymerase kappa (Pol κ) contributes to the replication checkpoint response and is required for recovery after replication stress. We found that Pol κ is implicated in the synthesis of short DNA intermediates at stalled forks, facilitating the recruitment of the 9-1-1 checkpoint clamp. Furthermore, we show that Pol κ interacts with the Rad9 subunit of the 9-1-1 complex. Finally, we show that this novel checkpoint function of Pol κ is required for the maintenance of genomic stability and cell proliferation in unstressed human cells.

Analysis of DNA Replication by Optical Mapping in Nanochannels.

DNA replication is essential to maintain genome integrity in S phase of the cell division cycle. Accumulation of stalled replication forks is a major source of genetic instability, and likely constitutes a key driver of tumorigenesis. The mechanisms of regulation of replication fork progression have therefore been extensively investigated, in particular with DNA combing, an optical mapping technique that allows the stretching of single molecules and the mapping of active region for DNA synthesis by fluorescence microscopy. DNA linearization in nanochannels has been successfully used to probe genomic information patterns along single chromosomes, and has been proposed to be a competitive alternative to DNA combing. Yet this conjecture remains to be confirmed experimentally. Here, two complementary techniques are established to detect the genomic distribution of tracks of newly synthesized DNA in human cells by optical mapping in nanochannels. Their respective advantages and limitations are compared, and applied them to detect deregulations of the replication program induced by the antitumor drug hydroxyurea. The developments here thus broaden the field of applications accessible to nanofluidic technologies, and can be used in the future as part for molecular diagnostics in the context of high throughput cancer drug screening.

Identification of the main barriers to Ku accumulation in chromatin.

Repair of DNA double strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Given its high affinity for ends, multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to ~1-2 molecules per DNA end. The mechanisms enforcing this limit are currently unknown. Here we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process which actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism which operates in S-phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together our data shed light on the multiple layers of coordinated mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions.

Cytidine Deaminase Resolves Replicative Stress and Protects Pancreatic Cancer from DNA-Targeting Drugs.

Cytidine deaminase (CDA) functions in the pyrimidine salvage pathway for DNA and RNA syntheses and has been shown to protect cancer cells from deoxycytidine-based chemotherapies. In this study, we observed that CDA was overexpressed in pancreatic adenocarcinoma from patients at baseline and was essential for experimental tumor growth. Mechanistic investigations revealed that CDA localized to replication forks where it increased replication speed, improved replication fork restart efficiency, reduced endogenous replication stress, minimized DNA breaks, and regulated genetic stability during DNA replication. In cellular pancreatic cancer models, high CDA expression correlated with resistance to DNA-damaging agents. Silencing CDA in patient-derived primary cultures in vitro and in orthotopic xenografts in vivo increased replication stress and sensitized pancreatic adenocarcinoma cells to oxaliplatin. This study sheds light on the role of CDA in pancreatic adenocarcinoma, offering insights into how this tumor type modulates replication stress. These findings suggest that CDA expression could potentially predict therapeutic efficacy and that targeting CDA induces intolerable levels of replication stress in cancer cells, particularly when combined with DNA-targeted therapies. SIGNIFICANCE: Cytidine deaminase reduces replication stress and regulates DNA replication to confer resistance to DNA-damaging drugs in pancreatic cancer, unveiling a molecular vulnerability that could enhance treatment response.

DNA polymerase theta up-regulation is associated with poor survival in breast cancer, perturbs DNA replication, and promotes genetic instability.

"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 (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.

RIP140 regulates POLK gene expression and the response to alkylating drugs in colon cancer cells.

Aim: The transcription factor RIP140 (receptor interacting protein of 140 kDa) is involved in intestinal tumorigenesis. It plays a role in the control of microsatellite instability (MSI), through the regulation of MSH2 and MSH6 gene expression. The aim of this study was to explore its effect on the expression of POLK, the gene encoding the specialized translesion synthesis (TLS) DNA polymerase κ known to perform accurate DNA synthesis at microsatellites. Methods: Different mouse models and engineered human colorectal cancer (CRC) cell lines were used to analyze by RT-qPCR, while Western blotting and luciferase assays were used to elucidate the role of RIP140 on POLK gene expression. Published DNA microarray datasets were reanalyzed. The in vitro sensitivity of CRC cells to methyl methane sulfonate and cisplatin was determined. Results: RIP140 positively regulates, at the transcriptional level, the expression of the POLK gene, and this effect involves, at least partly, the p53 tumor suppressor. In different cohorts of CRC biopsies (with or without MSI), a strong positive correlation was observed between RIP140 and POLK gene expression. In connection with its effect on POLK levels and the TLS function of this polymerase, the cellular response to methyl methane sulfonate was increased in cells lacking the Rip140 gene. Finally, the association of RIP140 expression with better overall survival of CRC patients was observed only when the corresponding tumors exhibited low levels of POLK, thus strengthening the functional link between the two genes in human CRC. Conclusion: The regulation of POLK gene expression by RIP140 could thus contribute to the maintenance of microsatellite stability, and more generally to the control of genome integrity.

Active site mutations in mammalian DNA polymerase delta alter accuracy and replication fork progression.

DNA polymerase δ (pol δ) is one of the two main replicative polymerases in eukaryotes; it synthesizes the lagging DNA strand and also functions in DNA repair. In previous work, we demonstrated that heterozygous expression of the pol δ L604G variant in mice results in normal life span and no apparent phenotype, whereas a different substitution at the same position, L604K, is associated with shortened life span and accelerated carcinogenesis. Here, we report in vitro analysis of the homologous mutations at position Leu-606 in human pol δ. Four-subunit human pol δ variants that harbor or lack 3' → 5'-exonucleolytic proofreading activity were purified from Escherichia coli. The pol δ L606G and L606K holoenzymes retain catalytic activity and processivity similar to that of wild type pol δ. pol δ L606G is highly error prone, incorporating single noncomplementary nucleotides at a high frequency during DNA synthesis, whereas pol δ L606K is extremely accurate, with a higher fidelity of single nucleotide incorporation by the active site than that of wild type pol δ. However, pol δ L606K is impaired in the bypass of DNA adducts, and the homologous variant in mouse embryonic fibroblasts results in a decreased rate of replication fork progression in vivo. These results indicate that different substitutions at a single active site residue in a eukaryotic polymerase can either increase or decrease the accuracy of synthesis relative to wild type and suggest that enhanced fidelity of base selection by a polymerase active site can result in impaired lesion bypass and delayed replication fork progression.

A Catalytically Independent Function of Human DNA Polymerase Kappa Controls the Stability and Abundance of Checkpoint Kinase 1.

DNA polymerase kappa (Pol κ) has been well documented thus far for its specialized DNA synthesis activity during translesion replication, progression of replication forks through regions difficult to replicate, restart of stalled forks, and replication checkpoint efficiency. Pol κ is also required for the stabilization of stalled forks, although the mechanisms are poorly understood. In this study, we unveiled an unexpected role for Pol κ in controlling the stability and abundance of checkpoint kinase 1 (Chk1), an important actor for the replication checkpoint and fork stabilization. We found that loss of Pol κ decreased the Chk1 protein level in the nuclei of four human cell lines. Pol κ and not the other Y family polymerase members is required to maintain the Chk1 protein pool all along the cell cycle. We showed that Pol κ depletion affected the protein stability of Chk1 and protected it from proteasome degradation. Importantly, we also observed that the fork restart defects observed in Pol κ-depleted cells could be overcome by the reexpression of Chk1. Strikingly, this new function of Pol κ does not require its catalytic activity. We propose that Pol κ could contribute to the protection of stalled forks through Chk1 stability.

KDM5A and KDM5B histone-demethylases contribute to HU-induced replication stress response and tolerance.

KDM5A and KDM5B histone-demethylases are overexpressed in many cancers and have been involved in drug tolerance. Here, we describe that KDM5A, together with KDM5B, contribute to replication stress (RS) response and tolerance. First, they positively regulate RRM2, the regulatory subunit of ribonucleotide reductase. Second, they are required for optimal levels of activated Chk1, a major player of the intra-S phase checkpoint that protects cells from RS. We also found that KDM5A is enriched at ongoing replication forks and associates with both PCNA and Chk1. Because RRM2 is a major determinant of replication stress tolerance, we developed cells resistant to HU, and show that KDM5A/B proteins are required for both RRM2 overexpression and tolerance to HU. Altogether, our results indicate that KDM5A/B are major players of RS management. They also show that drugs targeting the enzymatic activity of KDM5 proteins may not affect all cancer-related consequences of KDM5A/B overexpression.

Role of TLS DNA polymerases eta and kappa in processing naturally occurring structured DNA in human cells.

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, for example, 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 eta, pol iota, and pol kappa), specialized enzymes known to synthesize DNA through DNA damage. We show that depletion by RNA interference of expression of the genes for Pol eta or Pol kappa, but not Pol iota, sensitizes U2OS cells treated with the G4-tetraplex interactive compound telomestatin and triggers double-strand breaks in HeLa cells harboring 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 kappa 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 eta and pol kappa. We discuss these data in the light of their downregulation in human cancers.

Overexpression of Claspin and Timeless protects cancer cells from replication stress in a checkpoint-independent manner.

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.

Deregulated DNA polymerase beta induces chromosome instability and tumorigenesis.

To reach the biological alterations that characterize cancer, the genome of tumor cells must acquire increased mutability resulting from a malfunction of a network of genome stability systems, e.g., cell cycle arrest, DNA repair, and high accuracy of DNA synthesis during DNA replication. Numeric chromosomal imbalance, referred to as aneuploidy, is the most prevalent genetic changes recorded among many types of solid tumors. We report here that ectopic expression in cells of DNA polymerase beta, an error-prone enzyme frequently over-regulated in human tumors, induces aneuploidy, an abnormal localization of the centrosome-associated gamma-tubulin protein during mitosis, a deficient mitotic checkpoint, and promotes tumorigenesis in nude immunodeficient mice. Thus, we find that alteration of polymerase beta expression appears to induce major genetic changes associated with a malignant phenotype.

Cyclin Kinase-independent role of p21CDKN1A in the promotion of nascent DNA elongation in unstressed cells.

The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21's PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis.

Role of DNA polymerase κ in the maintenance of genomic stability.

To ensure high cell viability and genomic stability, cells have evolved two major mechanisms to deal with the constant challenge of DNA replication fork arrest during S phase of the cell cycle: (1) induction of the ataxia telangiectasia and Rad3-related (ATR) replication checkpoint mechanism, and (2) activation of a pathway that bypasses DNA damage and DNA with abnormal structure and is mediated by translesion synthesis (TLS) Y-family DNA polymerases. This review focuses on how DNA polymerase kappa (Pol κ), one of the most highly conserved TLS DNA polymerases, is involved in each of these pathways and thereby coordinates them to choreograph the response to a stalled replication fork. We also describe how loss of Pol κ regulation, which occurs frequently in human cancers, affects genomic stability and contributes to cancer development.

Expression of the microtubule-associated protein MAP9/ASAP and its partners AURKA and PLK1 in colorectal and breast cancers.

BACKGROUND: Colorectal and breast cancers are among the most common cancers worldwide. They result from a conjugated deficiency of gene maintenance and cell cycle control. OBJECTIVE: We investigate the expression of the microtubule-associated protein MAP9/ASAP and its two partners AURKA and PLK1 in colorectal tumors as well as in ductal breast cancers. MATERIALS AND METHODS: 26 colorectal cancer samples and adjacent normal tissues and 77 ductal breast cancer samples from grade I to grade III were collected. Real-time quantitative PCR was used to analyse the expression of MAP9, AURKA, and PLK1. Results. Expression of MAP9 is downregulated in colorectal cancer compared to normal tissues (P > 10(-3)), whereas those of AURKA and PLK1 are upregulated (P > 10(-4)). In ductal breast cancer, we found a grade-dependent increase of AURKA expression (P > 10(-3)), while the variations of expression of MAP9 and PLK1 are not significant (P > 0.2). CONCLUSIONS: MAP9 downregulation is associated with colorectal malignancy and could be used as a disease marker and a new drug target, while AURKA and PLK1 are upregulated. In ductal breast cancer, AURKA overexpression is strongly associated with the tumor grade and is therefore of prognostic value for the progression of the disease.

Upregulation of error-prone DNA polymerases beta and kappa slows down fork progression without activating the replication checkpoint.

There is rising evidence that cancer development is associated from its earliest stages with DNA replication stress, a major source of spontaneous genomic instability. However, the origin of these replication defects has remained unclear. We have investigated the consequences of upregulating error-prone DNA polymerases (pol) beta and kappa on chromosomal DNA replication. These enzymes are misregulated in different types of cancers and induce major chromosomal instabilities when overexpressed at low levels. Here, we have used DNA combing to show that a moderate overexpression of pol beta or pol kappa is sufficient to impede replication fork progression and to promote the activation of additional replication origins. Interestingly, alterations of the normal replication program induced by excess error-prone polymerases were not detected by the replication checkpoint. We therefore propose that upregulation of error-prone DNA polymerases induces a checkpoint-blind replication stress that contributes to genomic instability and to cancer development.

Xp38gamma/SAPK3 promotes meiotic G(2)/M transition in Xenopus oocytes and activates Cdc25C.

We have studied the role of p38 mitogen-activated protein kinases (MAPKs) in the meiotic maturation of Xenopus oocytes. Overexpression of a constitutively active mutant of the p38 activator MKK6 accelerates progesterone-induced maturation. Immunoprecipit ation experiments indicate that p38gamma/SAPK3 is the major p38 activated by MKK6 in the oocytes. We have cloned Xenopus p38gamma (Xp38gamma) and show that co-expression of active MKK6 with Xp38gamma induces oocyte maturation in the absence of progesterone. The maturation induced by Xp38gamma requires neither protein synthesis nor activation of the p42 MAPK-p90Rsk pathway, but it is blocked by cAMP-dependent protein kinase. A role for the endogenous Xp38gamma in progesterone-induced maturation is supported by the inhibitory effect of kinase-dead mutants of MKK6 and Xp38gamma. Furthermore, MKK6 can rescue the inhibition of oocyte maturation by anthrax lethal factor, a protease that inactivates MAPK kinases. We also show that Xp38gamma can activate the phosphatase XCdc25C, and we identified Ser205 of XCdc25C as a major phosphorylation site for Xp38gamma. Our results indicate that phosphorylation of XCdc25C by Xp38gamma/SAPK3 is important for the meiotic G(2)/M progression of Xenopus oocytes.