Participation of mouse DNA polymerase ι in strand-biased mutagenic bypass of UV photoproducts and suppression of skin cancer

Abstract

DNA polymerase (pol ) is a conserved Y family enzyme that is implicated in translesion DNA synthesis (TLS) but whose cellular functions remain uncertain. To test the hypothesis that pol performs TLS in cells, we compared UV-induced mutagenesis in primary fibroblasts derived from wild-type mice to mice lacking functional pol , pol , or both. A deficiency in mouse DNA polymerase (pol ) enhanced UV-induced Hprt mutant frequencies. This enhanced UV-induced mutagenesis and UV-induced mutagenesis in wild-type cells were strongly diminished in cells deficient in pol , indicating that pol participates in the bypass of UV photoproducts in cells. Moreover, a clear strand bias among UV-induced base substitutions was observed in wild-type cells that was diminished in pol -and pol -deficient mouse cells and abolished in cells deficient in both enzymes. These data suggest that these enzymes bypass UV photoproducts in an asymmetric manner. To determine whether pol status affects cancer susceptibility, we compared the UV-induced skin cancer susceptibility of wild-type mice to mice lacking functional pol , pol , or both. Although pol deficiency alone had no effect, UV-induced skin tumors in pol -deficient mice developed 4 weeks earlier in mice concomitantly deficient in pol . Collectively, these data reveal functions for pol in bypassing UV photoproducts and in delaying the onset of UV-induced skin cancer. The most compelling evidence for a role in translesion synthesis (TLS) is with pol . pol can efficiently bypass a cis-syn thymine-thymine dimer (6, 7), it localizes at replication foci after UV irradiation of mammalian cells (8), and its inactivation in XPV patients (9, 10) increases UV light-induced mutagenesis and skin cancer (reviewed in refs. 4 and 11). Increased UVinduced mutagenesis associated with pol deficiency indicates that mutagenic bypass of UV photoproducts can be catalyzed by other polymerases. One candidate is pol (2), which can bypass a thymine-thymine dimer in vitro (12) and is required for a large proportion of UV mutagenesis in vivo (2,13,14). Another candidate is pol , which physically interacts with pol and colocalizes with pol at replication foci after UV irradiation (8).Two hypotheses have been put forth regarding the outcome of putative UV photoproduct bypass by pol . One suggests that bypass should be mutagenic because of pol -catalyzed misinsertion of nucleotides opposite photoproducts (15, 16). pol is renowned for high rates of misinsertion of nucleotides opposite template pyrimidines during DNA synthesis in vitro (reviewed in ref.3). However, a study of UV-induced mutagenesis in cultured 293T cells in which pol expression was decreased by using siRNA concluded that pol has no significant role in UV lesion bypass and mutagenesis (17). A second hypothesis is that pol bypass could be antimutagenic for UV-induced C to T transition mutations. This antimutagenic hypothesis derives from a pol preference for inserting dGMP opposite template T, leading to the suggestion (18, 19) that synthesis by pol could b...

DNA polymerase ζ cooperates with polymerases κ and ι in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients

“…2B). Similarly, knocking-down the expression of POLI had no effect on UV sensitivity, which was different from several (but not all) previous reports (13)(14)(15)22). In contrast, knocking-down the expression of POLK increased UV sensitivity up to 3.5-5-fold, and REV3L by up to 33-to 39-fold (Fig.…”

“…reports (13)(14)(15)22). As far as mutagenesis is concerned, we saw essentially no effect on mutagenic TLS across a TT CPD when POLI was knocked-down.…”

“…As far as mutagenesis is concerned, we saw essentially no effect on mutagenic TLS across a TT CPD when POLI was knocked-down. Previous studies reported conflicting results of either decreased, altered or unchanged UV mutagenesis in cells deficient in pol (13)(14)(15)35). Like in the case of pol, this may reflect differences in methodologies or cell types.…”

“…The UV hypermutability is explained by the activity of a back-up DNA polymerase that performs TLS across CPD with lower efficiency and higher error-frequency. Although there is evidence that pol is involved in TLS across CPD in XPV cells (13)(14)(15), additional polymerases may be involved, and the picture is far from being complete. This is an important issue because these polymerases are likely to be driving sunlight-induced skin carcinogenesis in XPV patients.…”

DNA polymerase θ (POLQ) can extend from mismatches and from bases opposite a (6-4) photoproduct

“…It substantially distorts DNA and no single DNA polymerases in human cells can complete the translesion synthesis reaction alone. Both of the Y-family enzymes pol ι and pol η are involved in bypass and associated mutagenesis by UV radiation [26][27][28][29]. Pol ι is the only bypass DNA polymerase in human cells that correctly inserts an A opposite the first T of (6-4)PP; insertion opposite the second T of a (6-4)PP is errorprone [19].…”