ATR/Chk1 pathway is essential for resumption of DNA synthesis and cell survival in UV-irradiated XP variant cells

Centre Nationale de Recherche Scientifique (CNRS) UMR8200, Laboratoire Stabilité Génétique et Oncogenèse, Université Paris-Sud, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94800 Villejuif, France.
Human Molecular Genetics (Impact Factor: 6.68). 05/2010; 19(9):1690-701. DOI: 10.1093/hmg/ddq046
Source: PubMed

ABSTRACT 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.

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    • "Indeed, replication fork stalling at UV-induced DNA damage activates a checkpoint regulated by the ATR kinase that allows S-phase slow down and stabilization of replication forks (for review, see Cimprich & Cortez, 2008). We have previously shown that persistent inhibition of DNA synthesis in UV-irradiated cells deficient in Pol was associated with a stronger and persistent activation of ATR checkpoint (Despras et al, 2010). We analyzed the phosphorylation status of various ATR targets. "
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    ABSTRACT: 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. Copyright © 2015 Elsevier B.V. All rights reserved.
    DNA Repair 02/2015; 29. DOI:10.1016/j.dnarep.2015.02.017 · 3.36 Impact Factor
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    • "The identity of this band with Polη was demonstrated in wild-type cells transfected with a siRNA produced against the POLH gene. In this experiment, we used a unique siRNA because it has been already tested in our laboratory [Despras et al., 2010] and it is used only to show the disappearance of the protein band. As shown in Figure 2C, the 80 kDa band almost completely disappears in the siRNA-treated cells indicating we are looking to the real Polη protein. "
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    ABSTRACT: 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 XP-V 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, 6 for squamous-cell carcinoma and 5 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. This article is protected by copyright. All rights reserved.
    Human Mutation 01/2014; 35(1). DOI:10.1002/humu.22462 · 5.05 Impact Factor
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    • "How does the cell compartmentalize mutation rates within the genome during its duplication? In eukaryotes, the intra-S phase checkpoint pathway (ATR-Chk1) mediates between DNA replication and DNA repair and slows or stops replication when DNA damage occurs (Despras et al. 2010). During normal S phase, the checkpoint system also participates in mediating the rate at which DNA replication origins fire, and hence influences the rate of progression through S phase (Herrick 2010). "
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    ABSTRACT: Growing evidence suggests that many vertebrate lineages are evolving at significantly different rates. As a first approximation of evolutionary rates, we assessed the amount of neutral (dS) and non-neutral (dN) substitutions that have accumulated within and across sister clades since the time of their divergence. We found that in fish, tetraodontiformes (pufferfish) are evolving at faster rates than cypriniformes (fresh water teleosts), while cypriniformes are evolving faster than elasmobranchs (sharks, skates and rays). A similar rate variation was observed in salamanders: plethodontidae were found to evolve at a rate nearly two fold faster than larger genome paedomorphs. We discuss possible explanations for this striking variation in substitution rates among different vertebrate lineages that occupy widely diverse habitats and niches.
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