期刊
DNA REPAIR
卷 35, 期 -, 页码 1-12出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.dnarep.2015.07.002
关键词
Y-family DNA polymerase; Translesion DNA synthesis; Ribonucleotide excision repair; DNA polymerase eta; Cyclobutane pyrimidine timer; RNase H2
资金
- National Institute of Child Health and Human Development/National Institutes of Health Intramural Research Program
- National Institute of Child Health and Human Development Intramural Research Program
DNA polymerase eta (pol eta) is best characterized for its ability to perform accurate and efficient translesion DNA synthesis (TLS) through cyclobutane pyrimidine dimers (CPDs). To ensure accurate bypass the polymerase is not only required to select the correct base, but also discriminate between NTPs and dNTPs. Most DNA polymerases have a conserved steric gate residue which functions to prevent incorporation of NMPs during DNA synthesis. Here, we demonstrate that the Phe35 residue of Saccharomyces cerevisiae pol eta functions as a steric gate to limit the use of ribonucleotides during polymerization both in vitro and in vivo. Unlike the related pol eta enzyme, wild-type pol eta does not readily incorporate NMPs in vitro. In contrast, a pol eta F35A mutant incorporates NMPs on both damaged and undamaged DNA in vitro with a high degree of base selectivity. An S.cerevisiae strain expressing pol eta F35A (rad30-F35A) that is also deficient for nucleotide excision repair (rad1 Delta) and the TLS polymerase, pol zeta (rev3 Delta), is extremely sensitive to UV-light. The sensitivity is due, in part, to RNase H2 activity, as an isogenic rnh201 Delta strain is roughly 50-fold more UV-resistant than its RNH201(+) counterpart. Interestingly the rad1 Delta rev3 Delta rad30-F35A rnh201 Delta strain exhibits a significant increase in the extent of spontaneous mutagenesis with a spectrum dominated by 1 bp deletions at runs of template Ts. We hypothesize that the increased mutagenesis is due to rA incorporation at these sites and that the short poly rA tract is subsequently repaired in an error-prone manner by a novel repair pathway that is specifically targeted to polyribonucleotide tracks. These data indicate that under certain conditions, pol eta can compete with the cell's replicases and gain access to undamaged genomic DNA. Such observations are consistent with a role for pol eta in replicating common fragile sites (CFS) in human cells. Published by Elsevier B.V.
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