Abstract
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.
Highlights
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]
We found that mutation of either ubiquitin binding zinc finger motifs (UBZs) or proliferating cell nuclear antigen (PCNA) binding peptide (PIP) in polymerases kappa (Polk) did not abolish the interaction, suggesting that the UBZs and PIP domains are not essential for the interaction
We observed that Polk and MSH2 co-localized at nuclear foci after exposure of cells to UV radiation (Figure 1C)
Summary
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]. 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 [8,9,10]. Increased spontaneous mutagenesis has been observed in some tissues from Polk-deficient mice [9], Nucleic Acids Research, 2013, Vol 41, No 22 10313 suggesting that some, if not all TLS polymerases, are required for the maintenance of genome stability
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