Effects of Accessory Proteins on the Bypass of a cis-syn Thymine−Thymine Dimer by Saccharomyces cerevisiae DNA Polymerase η

Abstract

Among several hypotheses to explain how translesion synthesis (TLS) by DNA polymerase η (pol η) suppresses ultraviolet light-induced mutagenesis in vivo despite the fact that pol η copies DNA with low fidelity, here we test whether replication accessory proteins enhance the fidelity of TLS by pol η. We first show that the single-stranded DNA binding protein RPA, the sliding clamp PCNA, and the clamp loader RFC slightly increase the processivity of yeast pol η and its ability to recycle to new template primers. However, these increases are small, and they are similar when copying an undamaged template and a template containing a cis-syn TT dimer. Consequently, the accessory proteins do not strongly stimulate the already robust TT dimer bypass efficiency of pol η. We then perform a comprehensive analysis of yeast pol η fidelity. We show that it is much less accurate than other yeast DNA polymerases and that the accessory proteins have little effect on fidelity when copying undamaged templates or when bypassing a TT dimer. Thus, although accessory proteins clearly participate in pol η functions in vivo, they do not appear to help suppress UV mutagenesis by improving pol η bypass fidelity per se.Translesion synthesis (TLS 1 ) is one mechanism of damage tolerance employed by cells when synthesis by the major replicative polymerases is blocked by lesions. Several polymerases in different families have been shown to bypass lesions in vitro (see ref 1 and references therein). Of these, the role of pol η in the bypass of UV photoproducts is the best understood. In humans, the loss of pol η causes the variant (XP-V) form of xeroderma pigmentosum (2,3), a disease characterized by greatly increased susceptibility to sunlight-induced skin cancer (see refs 4 and 5 and references therein). A key property of human and yeast cells lacking pol η is increased mutagenesis following exposure to ultraviolet light (6-13). A key property of human and yeast pol η is its ability to bypass cyclobutane pyrimidine dimers (CPDs) with much higher efficiency than that of other eukaryotic DNA polymerases (14-18). These facts imply that pol η participates in the bypass of slowly repaired cyclobutane pyrimidine dimers in a manner that avoids mutations such that in its absence, other polymerases perform mutagenic bypass that ultimately results in skin cancer. Consistent with the participation of pol η in CPD bypass that avoids mutations are seminal studies of single nucleotide insertion (17,19,20), demonstrating that yeast and human pol η preferentially insert dAMP opposite both template thymines of a cis-syn thymine-thymine dimer (TTD). This selectivity for inserting a correct nucleotide undoubtedly contributes to CPD bypass in cells that reduces UV-induced mutagenesis in humans, mice, and yeast. The actual rate of base substitutions generated per CPD bypass event in human cells is unknown. Fortunately, quantitative in vivo measurements in S. cerevisiae are available and indicate that bypass of TC and CC dimers generates less than one bas...

The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases

“…Subsequent studies have determined that the fidelity for TTD bypass by yeast and human pol η is actually quite low, e.g., one dGMP incorporated opposite the 3′-T of the dimer for ~30 dAMP incorporations. This high error rate is similar to that observed when pol η copies the corresponding undamaged thymine [91,92]. The fidelity of homologous Sso Dpo4 is even lower (approaching 1 error in every 10 bypass events), and in this case the error rate is far higher than for copying the equivalent undamaged base [91].…”

“…Again, however, there is currently no evidence that Rev1/polymerase interactions alter the fidelity of TLS. Thus, the few studies that have been carried out so far come to the same conclusions as the results with the major replicative polymerases, namely that the polymerases themselves are the prime determinants of the fidelity of TLS [92,111].…”

The Polymerase η Translesion Synthesis DNA Polymerase Acts Independently of the Mismatch Repair System To Limit Mutagenesis Caused by 7,8-Dihydro-8-Oxoguanine in Yeast

“…It thus requires that the PIP domain of Pol be intact (Table 1) (16,54) and may involve a specific interaction with the monoubiquitinated form of PCNA (39,54,58,63). Aside from this specific scenario, however, it is difficult to imagine a more global involvement of Pol in MMR; the relatively low fidelity of this enzyme on undamaged DNA would lead to many nonspecific errors during MMRassociated gap filling (35). Indeed, the only unequivocal evidence of Pol functioning in MMR is in the specialized somatic hypermutation of B cells (9).…”

“…At GO lesions, Pol is 10-fold more accurate and efficient than Pol␦ (34). When given an undamaged DNA template, however, the base substitution error frequency of Pol in vitro is 3 orders of magnitude greater than that of a typical replicative polymerase (35). In addition to Pol, there are two other TLS polymerases in S. cerevisiae (Pol and Rev1), but neither has been implicated in the bypass of GO lesions (10,48).…”

Effect of pH on the Misincorporation Rate of DNA Polymerase η

“…32) In contrast to DNA polymerase η, DNA polymerase α did not show a drastic change in the misincorporation rate of dGTP opposite template T with pH (Figs. 4, 5).…”