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National Institute of Environmental Health Sciences

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Publication Detail

Title: Biochemical analysis of DNA polymerase η fidelity in the presence of replication protein A.

Authors: Suarez, Samuel C; Toffton, Shannon M; McCulloch, Scott D

Published In PLoS One, (2014)

Abstract: DNA polymerase η (pol η) synthesizes across from damaged DNA templates in order to prevent deleterious consequences like replication fork collapse and double-strand breaks. This process, termed translesion synthesis (TLS), is an overall positive for the cell, as cells deficient in pol η display higher mutation rates. This outcome occurs despite the fact that the in vitro fidelity of bypass by pol η alone is moderate to low, depending on the lesion being copied. One possible means of increasing the fidelity of pol η is interaction with replication accessory proteins present at the replication fork. We have previously utilized a bacteriophage based screening system to measure the fidelity of bypass using purified proteins. Here we report on the fidelity effects of a single stranded binding protein, replication protein A (RPA), when copying the oxidative lesion 7,8-dihydro-8-oxo-guanine(8-oxoG) and the UV-induced cis-syn thymine-thymine cyclobutane pyrimidine dimer (T-T CPD). We observed no change in fidelity dependent on RPA when copying these damaged templates. This result is consistent in multiple position contexts. We previously identified single amino acid substitution mutants of pol η that have specific effects on fidelity when copying both damaged and undamaged templates. In order to confirm our results, we examined the Q38A and Y52E mutants in the same full-length construct. We again observed no difference when RPA was added to the bypass reaction, with the mutant forms of pol η displaying similar fidelity regardless of RPA status. We do, however, observe some slight effects when copying undamaged DNA, similar to those we have described previously. Our results indicate that RPA by itself does not affect pol η dependent lesion bypass fidelity when copying either 8-oxoG or T-T CPD lesions.

PubMed ID: 24824831 Exiting the NIEHS site

MeSH Terms: DNA Damage/genetics*; DNA Primers/genetics; DNA Repair/physiology*; DNA Replication/physiology*; DNA-Directed DNA Polymerase/metabolism*; Guanine/analogs & derivatives; Guanine/metabolism; Humans; Mutation, Missense/genetics; Pyrimidine Dimers/metabolism; Recombinant Proteins/genetics; Replication Protein A/metabolism*

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