Unlocking the steric gate of DNA polymerase η leads to increased genomic instability in Saccharomyces cerevisiae.
DNA polymerase η (pol η) 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 η functions as a steric gate to limit the use of ribonucleotides during polymerization both in vitro and in vivo. Unlike the related pol ι enzyme, wild-type pol η does not readily incorporate NMPs in vitro. In contrast, a pol η 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 η F35A (rad30-F35A) that is also deficient for nucleotide excision repair (rad1Δ) and the TLS polymerase, pol ζ (rev3Δ), is extremely sensitive to UV-light. The sensitivity is due, in part, to RNase H2 activity, as an isogenic rnh201Δ strain is roughly 50-fold more UV-resistant than its RNH201(+) counterpart. Interestingly the rad1Δ rev3Δ rad30-F35A rnh201Δ strain exhibits a significant increase in the extent of spontaneous mutagenesis with a spectrum dominated by 1bp 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 η can compete with the cell's replicases and gain access to undamaged genomic DNA. Such observations are consistent with a role for pol η in replicating common fragile sites (CFS) in human cells.
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.
Katherine A
KA
Donigan
Program in Genomics of Differentiation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Susana M
SM
Cerritelli
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.
John P
JP
McDonald
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.
Alexandra
A
Vaisman
Program in Genomics of Differentiation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Robert J
RJ
Crouch
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA. Electronic address: woodgate@nih.gov.
Roger
R
Woodgate
D000409:Alanine / Q000737:chemistry / Q000235:genetics; D019943:Amino Acid Substitution; D001483:Base Sequence; D017124:Conserved Sequence; D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004261:DNA Replication; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D042822:Genomic Instability; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D011131:Polyribonucleotides / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
D000409:Alanine; D004271:DNA, Fungal; D004259:DNA-Directed DNA Polymerase; D010649:Phenylalanine; D011131:Polyribonucleotides; D012265:Ribonucleotides; D029701:Saccharomyces cerevisiae Proteins; C023140:DNA replicase; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases
Cyclobutane pyrimidine dimer; DNA polymerase η; RNase H2; Ribonucleotide excision repair; Translesion DNA synthesis; Y-family DNA polymerase