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Unlocking the steric gate of DNA polymerase ? leads to increased genomic instability in Saccharomyces cerevisiae.

ABSTRACT: DNA polymerase ? (pol ?) 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 ? functions as a steric gate to limit the use of ribonucleotides during polymerization both in vitro and in vivo. Unlike the related pol ? enzyme, wild-type pol ? does not readily incorporate NMPs in vitro. In contrast, a pol ? 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 ? F35A (rad30-F35A) that is also deficient for nucleotide excision repair (rad1?) and the TLS polymerase, pol ? (rev3?), is extremely sensitive to UV-light. The sensitivity is due, in part, to RNase H2 activity, as an isogenic rnh201? strain is roughly 50-fold more UV-resistant than its RNH201(+) counterpart. Interestingly the rad1? rev3? rad30-F35A rnh201? 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 ? can compete with the cell's replicases and gain access to undamaged genomic DNA. Such observations are consistent with a role for pol ? in replicating common fragile sites (CFS) in human cells.

SUBMITTER: Donigan KA

PROVIDER: S-EPMC4651834 | biostudies-other | 2015 Nov

REPOSITORIES: biostudies-other

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Unlocking the steric gate of DNA polymerase η leads to increased genomic instability in Saccharomyces cerevisiae.

Donigan Katherine A KA Cerritelli Susana M SM McDonald John P JP Vaisman Alexandra A Crouch Robert J RJ Woodgate Roger R

DNA repair 20150807

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 Saccharomyc ...[more]

PMID: 26340535

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Project description:How microbial cells leverage their phenotypic potential to survive in a changing environment is a complex biological problem, with important implications for pathogenesis and species evolution. Stochastic phenotype switching, a particularly fascinating adaptive approach observed in numerous species across the tree of life, introduces phenotypic diversity into a population through mechanisms which have remained difficult to define. Here we describe our investigations into the mechanistic basis of colony morphology phenotype switching which occurs in populations of a pathogenic isolate of Saccharomyces cerevisiae, YJM311. We observed that clonal populations of YJM311 cells produce variant colonies that display altered morphologies and, using whole genome sequence analysis, discovered that these variant clones harbored an exceptional collection of karyotypes newly altered by de novo structural genomic variations (SVs). Overall, our analyses indicate that copy number alterations, more often than changes in allelic identity, provide the causative basis of this phenotypic variation. Individual variants carried between 1 and 16 de novo copy number variations, most of which were whole chromosomal aneuploidies. Notably, we found that the inherent stability of the diploid YJM311 genome is comparable to that of domesticated laboratory strains, indicating that the collections of SVs harbored by variant clones did not arise by a chronic chromosomal instability (CIN) mechanism. Rather, our data indicate that these variant clones acquired such complex karyotypic configurations simultaneously, during stochastic and transient episodes of punctuated systemic genomic instability (PSGI). Surprisingly, we found that the majority of these highly altered variant karyotypes were propagated with perfect fidelity in long-term passaging experiments, demonstrating that high aneuploidy burdens can often be conducive with prolonged genomic integrity. Together, our results demonstrate that colony morphology switching in YJM311 is driven by a stochastic process in which genome stability and plasticity are integrally coupled to phenotypic heterogeneity. Consequently, this system simultaneously introduces both phenotypic and genomic variation into a population of cells, which can, in turn perpetuate population diversity for many generations thereafter.

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Dataset Information

Unlocking the steric gate of DNA polymerase ? leads to increased genomic instability in Saccharomyces cerevisiae.

Publications

Unlocking the steric gate of DNA polymerase η leads to increased genomic instability in Saccharomyces cerevisiae.

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