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

Nucleic Acids Research, Dec 2013

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.

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

Lingna Lv 2 Fengli Wang 1 Xiaolu Ma 2 Yeran Yang 2 Zhifeng Wang 2 Hongmei Liu 1 Xiaoling Li 1 Zhenbo Liu 2 Ting Zhang 2 Min Huang 2 Errol C. Friedberg 0 Tie-Shan Tang 1 Caixia Guo 2 0 Department of Pathology, University of Texas Southwestern Medical Center , Dallas, TX 75390, USA 1 State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences , Beijing 100101, China 2 Laboratory of Cancer Genomics and Individualized Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences , Beijing 100101, China - 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 (Poli) 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 Poli and other TLS polymerases after UV irradiation of cells. Interestingly, expression of MSH2 in Rad18-deficient cells increased UV-induced Poli 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. INTRODUCTION Translesion DNA synthesis (TLS) is a mode of DNA damage tolerance that uses specialized DNA polymerases to support DNA synthesis past a spectrum of template strand base damage, thereby preventing stalled replication forks from collapse and possible cell death (1). Ten different specialized DNA polymerases in mammalian cells have been shown to support TLS in vitro. These enzymes are devoid of 30!50 proofreading exonuclease activity and replicate undamaged DNA in vitro with low fidelity and weak processivity (2). Among them, DNA polymerases kappa (Polk), iota (Poli), eta (PolZ) and REV1 belong to a novel DNA polymerase family (the Y-family) (3,4). Each of the Y-family polymerases exhibits a preference for the replicative bypass of specific types of base damage in DNA. For example, Polk and PolZ support accurate bypass of benzo[a]pyrene diol epoxide guanine adducts (B[a]P-dG) and solar ultraviolet (UV) radiation-induced cissyn thyminethymine cyclobutanepyrimidine dimers (CPDs), respectively (2,5,6). PolZ-deficient cells manifest UV radiation-induced mutagenesis, and a markedly elevated predisposition to UV radiation-induced skin cancer has been observed in PolZ-deficient humans and mice (3,4,7). Similarly, Polk-deficient mouse embryonic fibroblasts and embryonic stem cells are sensitive to the killing effects of benzo[a]pyrene, and exhibit enhanced benzo[a]pyrene diol epoxide-induced mutagenesis (810). Additionally, increased spontaneous mutagenesis has been observed in some tissues from Polk-deficient mice (9), suggesting that some, if not all TLS polymerases, are required for the maintenance of genome stability. These observations notwithstanding, over-expression of some specialized DNA polymerases results in elevated genomic instability that manifests with enhanced mutation rates as well as DNA strand breaks (3,11,12). Given the low fidelity of TLS polymerases copying undamaged templates in vitro, it is believed that TLS processes in vivo are strictly regulated to keep TLS polymerases mainly functioning at their cognate substrates in an error-free fashion. Consistent with these observations, dysregulation of Polk recruitment to replication forks promotes genomic instability (13). TLS in mammalian cells is promoted by monoubiquitination of proliferating cell nuclear antigen (PCNA). A number of studies have shown that monoubiquitinated PCNA exhibits enhanced interaction with PolZ, Polk, Poli and REV1, relative to unmodified PCNA (1419). In response to UV radiation, PCNA is monoubiquitinated at Lys164 by the ubiquitinconjugating enzyme Rad6 and its cognate ubiquitin ligase Rad18 (20,21). The upstream signal that activates PCNA monoubiquitination (PCNA-mUb) in vivo is replication protein A (RPA)-coated single-stranded DNA (ssDNA) at sites of stalled forks, in which RPA targets Rad18 to its sites of action (22). Monoubiquitinated PCNA is deubiquitinated primarily by the u (...truncated)


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Lingna Lv, Fengli Wang, Xiaolu Ma, Yeran Yang, Zhifeng Wang, Hongmei Liu, Xiaoling Li, Zhenbo Liu, Ting Zhang, Min Huang, Errol C. Friedberg, Tie-Shan Tang, Caixia Guo. Mismatch repair protein MSH2 regulates translesion DNA synthesis following exposure of cells to UV radiation, Nucleic Acids Research, 2013, pp. 10312-10322, 41/22, DOI: 10.1093/nar/gkt793