René M Overmeer

Leiden University Medical Centre, Leiden, South Holland, Netherlands

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Publications (6)49.35 Total impact

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    ABSTRACT: A network of DNA damage surveillance systems is triggered by sensing of DNA lesions and the initiation of a signal transduction cascade that activates genome-protection pathways including nucleotide excision repair (NER). NER operates through coordinated assembly of repair factors into pre- and post-incision complexes. Recent work identifies RPA as a key regulator of the transition from dual incision to repair-synthesis in UV-irradiated non-cycling cells, thereby averting the generation of unprocessed repair intermediates. These intermediates could lead to recombinogenic events and trigger a persistent ATR-dependent checkpoint signaling. It is now evident that DNA damage signaling is not limited to NER proficient cells. ATR-dependent checkpoint activation also occurs in UV-exposed non-cycling repair deficient cells coinciding with the formation of endonuclease APE1-mediated DNA strand breaks. In addition, the encounter of elongating RNA polymerase II (RNAPIIo) with DNA damage lesions and its persistent stalling provides a strong DNA damage signaling leading to cell cycle arrest, apoptosis and increased mutagenesis. The mechanism underlying the strong and strand specific induction of UV-induced mutations in NER deficient cells has been recently resolved by the finding that gene transcription itself increases UV-induced mutagenesis in a strand specific manner via increased deamination of cytosines. The cell removes the RNAPIIo-blocking DNA lesions by transcription-coupled repair (TC-NER) without displacement of the DNA damage stalled RNAPIIo. Deficiency in TC-NER associates with mutations in the CSA and CSB genes giving rise to the rare human disorder Cockayne syndrome (CS). CSB functions as a repair coupling factor to attract NER proteins, chromatin remodelers and the CSA-E3-ubiquitin ligase complex to the stalled RNAPIIo; CSA is dispensable for attraction of NER proteins, yet in cooperation with CSB is required to recruit XAB2, the nucleosomal binding protein HMGN1 and TFIIS. The molecular mechanisms by which these proteins bring about efficient TC-NER and trigger signaling after transcription arrest remain elusive; particularly the role of chromatin remodeling in TC-NER needs to be clarified in the context of anticipated structural changes that allow repair and transcription restart.
    DNA repair 05/2011; 10(7):743-50. · 4.20 Impact Factor
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    ABSTRACT: Activation of signaling pathways by UV radiation is a key event in the DNA damage response and initiated by different cellular processes. Here we show that non-cycling cells proficient in nucleotide excision repair (NER) initiate a rapid but transient activation of the damage response proteins p53 and H2AX; by contrast, NER-deficient cells display delayed but persistent signaling and inhibition of cell cycle progression upon release from G0 phase. In the absence of repair, UV-induced checkpoint activation coincides with the formation of single-strand DNA breaks by the action of the endonuclease Ape1. Although temporally distinct, activation of checkpoint proteins in NER-proficient and NER-deficient cells depends on a common pathway involving the ATR kinase. These data reveal that damage signaling in non-dividing cells proceeds via NER-dependent and NER-independent processing of UV photolesions through generation of DNA strand breaks, ultimately preventing the transition from G1 to S phase.
    Journal of Cell Science 02/2011; 124(Pt 3):435-46. · 5.88 Impact Factor
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    ABSTRACT: Single-stranded DNA gaps that might arise by futile repair processes can lead to mutagenic events and challenge genome integrity. Nucleotide excision repair (NER) is an evolutionarily conserved repair mechanism, essential for removal of helix-distorting DNA lesions. In the currently prevailing model, NER operates through coordinated assembly of repair factors into pre- and post-incision complexes; however, its regulation in vivo is poorly understood. Notably, the transition from dual incision to repair synthesis should be rigidly synchronized as it might lead to accumulation of unprocessed repair intermediates. We monitored NER regulatory events in vivo using sequential UV irradiations. Under conditions that allow incision yet prevent completion of repair synthesis or ligation, preincision factors can reassociate with new damage sites. In contrast, replication protein A remains at the incomplete NER sites and regulates a feedback loop from completion of DNA repair synthesis to subsequent damage recognition, independently of ATR signaling. Our data reveal an important function for replication protein A in averting further generation of DNA strand breaks that could lead to mutagenic and recombinogenic events.
    The Journal of Cell Biology 02/2011; 192(3):401-15. · 10.82 Impact Factor
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    ABSTRACT: Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3' termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5' phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA.
    Molecular and Cellular Biology 10/2010; 30(20):4828-39. · 5.04 Impact Factor
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    ABSTRACT: Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both pol kappa and pol delta, and both polymerases can be recovered in the same repair complexes. Pol kappa is recruited to repair sites by ubiquitinated PCNA and XRCC1 and pol delta by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on pol epsilon, recruitment of which is dependent on the alternative clamp loader CTF18-RFC.
    Molecular cell 03/2010; 37(5):714-27. · 14.61 Impact Factor
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    ABSTRACT: Hypoxia-inducible factors (HIFs) are critical transcription factors that mediate cell survival during reduced oxygen conditions (hypoxia). At regular oxygen conditions (normoxia), HIF-1alpha and HIF-2alpha are continuously synthesized in cells and degraded via the ubiquitin-proteasome pathway. During hypoxia, these proteins are stabilized and translocate to the nucleus to activate transcription of target genes that enable cell survival at reduced oxygen levels. HIF proteins are tightly regulated via post-translational modifications including phosphorylation, acetylation, prolyl-hydroxylation and ubiquitination. Here we show for the first time that exogenous and endogenous HIF-2alpha are also regulated via the ubiquitin-like modifier small ubiquitin-like modifiers (SUMO). Using mutational analysis, we found that K394, which is situated in the sumoylation consensus site LKEE, is the major SUMO acceptor site in HIF-2alpha. Functionally, sumoylation reduced the transcriptional activity of HIF-2alpha. Similar to HIF-1alpha, HIF-2alpha is regulated by the SUMO protease SENP1. The proteasome inhibitor MG132 strongly stabilized SUMO-2-conjugated HIF-2alpha during hypoxia but did not affect the total level of HIF-2alpha. The ubiquitin E3 ligases von Hippel-Lindau and RNF4 control the levels of sumoylated HIF-2alpha, indicating that sumoylated HIF-2alpha is degraded via SUMO-targeted ubiquitin ligases.
    Nucleic Acids Research 12/2009; 38(6):1922-31. · 8.81 Impact Factor