Regulation Of DNA Polymerase Kappa In Translesion DNA Synthesis And Its Novel Function In DNA Damage Response

Translesion DNA synthesis (TLS) can limit the production of DNA double-strand breaks and cell death due to collapse of replication forks by utilizing specialized DNA polymerases which have low fidelity to insert and/or extend nucleotides across the lesions. Given their low fidelity when copy undamaged templates in vitro, it is believed that in vivo TLS process is strictly regulated and over-activated TLS might lead to increased genome mutations through error-prone bypass pathway. It has been reported that TLS polymerases can bind with monoubiquitinated PCNA and thus be recruited to stalled replication forks to perform TLS functions. It has been found recently that several proteins regulate this process, including INO80, Spartan and BRCAl.To explore other possible factors engaging in UV-induced TLS, we immunoprecipitated DNA polymerase kappa (Polκ) from cell lysates and surprisingly found through mass spectrometry that TLS polymerase Polκ interacted with MSH2, an important mismatch repair protein. Knocking down of MSH2would impair PCNA monoubiquitination and the recruitment of Polκ to stalled replication forks after UV irradiation. Overexpressing MSH2in RAD18-/-(RAD18is a E3ligase, monoubiquitinating PCNA in response to various damage agents) cells would increase Polk foci at UV-induced damage sites without obvious changes on PCNA level. Further study found that MSH2was required for gap filling opposite genomic CPD lesions, which indicated that MSH2has a novel role in UV-induced DNA damage response.Besides, a number of studies suggest that some specialized TLS polymerases also support other aspects of DNA metabolism beyond TLS in vivo. For example, Polηcan synthesize DNA from D-loop recombination intermediates when an invading DNA strand serves as the primer. Polιhas also been reported to have functions in base excision repair (BER). Polk has been implicated in repair synthesis of DNA during nucleotide excision repair (NER) under some conditions. Here we show that mouse Polk could accumulate at laser-induced sites of damage in vivo resembling polymerases η and ι. The recruitment was mediated through Polk C-terminus which contains the PCNA-interactingpeptide, ubiquitin zinc finger motif2and nuclear localization signal. Interestingly, this recruitment was significantly reduced in MSH2-deficient LoVo cells and Rad18-depleted cells. We further observed that PolK-deficient mouse embryo fibroblasts were abnormally sensitive to H2O2treatment and displayed defects in both single-strand break repair and double-strand break repair. We speculate that Polk may have an important role in strand break repair following oxidative stress in vivo.These results will not only help us understand the underlying mechanisms about the newly identified noncanonical mismatch repair-induced genome instability, but also manifest the complicated crosstalk between TLS and other DNA damage response passways.We believe that these results will definitely provide clues to prevent carcinogenesis and enhance tumor killing through decreasing the mutagenesis of tumor cells before and during tumor therapy.