The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins
ABSTRACT DNA polymerase zeta (pol zeta) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol zeta alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol zeta. Pol zeta is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity of pol zeta is substantially lower than that of homologous B family pols alpha, delta and epsilon. Pol zeta is particularly error prone for substitutions in specific sequence contexts and generates multiple single base errors clustered in short patches at a rate that is unprecedented in comparison with other polymerases. The unique error specificity of pol zeta in vitro is consistent with Pol zeta-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex mutations observed here, indicates that pol zeta contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending them.
SourceAvailable from: Stanislaw K Jozwiakowski[Show abstract] [Hide abstract]
ABSTRACT: PrimPol is a recently identified polymerase involved in eukaryotic DNA damage tolerance, employed in both re-priming and translesion synthesis mechanisms to bypass nuclear and mitochondrial DNA lesions. In this report, we investigate how the enzymatic activities of human PrimPol are regulated. We show that, unlike other TLS polymerases, PrimPol is not stimulated by PCNA and does not interact with it in vivo. We identify that PrimPol interacts with both of the major single-strand binding proteins, RPA and mtSSB in vivo. Using NMR spectroscopy, we characterize the domains responsible for the PrimPol-RPA interaction, revealing that PrimPol binds directly to the N-terminal domain of RPA70. In contrast to the established role of SSBs in stimulating replicative polymerases, we find that SSBs significantly limit the primase and polymerase activities of PrimPol. To identify the requirement for this regulation, we employed two forward mutation assays to characterize PrimPol's replication fidelity. We find that PrimPol is a mutagenic polymerase, with a unique error specificity that is highly biased towards insertion-deletion errors. Given the error-prone disposition of PrimPol, we propose a mechanism whereby SSBs greatly restrict the contribution of this enzyme to DNA replication at stalled forks, thus reducing the mutagenic potential of PrimPol during genome replication.Nucleic Acids Research 12/2014; 43(2). DOI:10.1093/nar/gku1321 · 8.81 Impact Factor
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ABSTRACT: Translesion synthesis (TLS) helps cells to accomplish chromosomal replication in the presence of unrepaired DNA lesions. In eukaryotes, the bypass of most lesions involves a nucleotide insertion opposite the lesion by either a replicative or a specialized DNA polymerase, followed by extension of the resulting distorted primer terminus by DNA polymerase ζ (Polζ). The subsequent events leading to disengagement of the error-prone Polζ from the primer terminus and its replacement with an accurate replicative DNA polymerase remain largely unknown. As a first step toward understanding these events, we aimed to determine the length of DNA stretches synthesized in an error-prone manner during the Polζ-dependent lesion bypass. We developed new in vivo assays to identify the products of mutagenic TLS through a plasmid-borne tetrahydrofuran lesion and a UV-induced chromosomal lesion. We then surveyed the region downstream of the lesion site (in respect to the direction of TLS) for the presence of mutations indicative of an error-prone polymerase activity. The bypass of both lesions was associated with an approximately 300,000-fold increase in the mutation rate in the adjacent DNA segment, in comparison to the mutation rate during normal replication. The hypermutated tract extended 200 bp from the lesion in the plasmid-based assay and as far as 1 kb from the lesion in the chromosome-based assay. The mutation rate in this region was similar to the rate of errors produced by purified Polζ during copying of undamaged DNA in vitro. Further, no mutations downstream of the lesion were observed in rare TLS products recovered from Polζ-deficient cells. This led us to conclude that error-prone Polζ synthesis continues for several hundred nucleotides after the lesion bypass is completed. These results provide insight into the late steps of TLS and show that error-prone TLS tracts span a substantially larger region than previously appreciated.PLoS Genetics 03/2015; 11(3):e1005110. DOI:10.1371/journal.pgen.1005110 · 8.17 Impact Factor
Carrie M Stith