Dataset Information

Noncognate DNA damage prevents the formation of the active conformation of the Y-family DNA polymerases DinB and DNA polymerase ?.

ABSTRACT: Y-family DNA polymerases are specialized to copy damaged DNA, and are associated with increased mutagenesis, owing to their low fidelity. It is believed that the mechanism of nucleotide selection by Y-family DNA polymerases involves conformational changes preceding nucleotidyl transfer, but there is limited experimental evidence for such structural changes. In particular, nucleotide-induced conformational changes in bacterial or eukaryotic Y-family DNA polymerases have, to date, not been extensively characterized. Using hydrogen-deuterium exchange mass spectrometry, we demonstrate here that the Escherichia coli Y-family DNA polymerase DinB and its human ortholog DNA polymerase ? undergo a conserved nucleotide-induced conformational change in the presence of undamaged DNA and the correct incoming nucleotide. Notably, this holds true for damaged DNA containing N(2) -furfuryl-deoxyguanosine, which is efficiently copied by these two polymerases, but not for damaged DNA containing the major groove modification O(6) -methyl-deoxyguanosine, which is a poor substrate. Our observations suggest that DinB and DNA polymerase ? utilize a common mechanism for nucleotide selection involving a conserved prechemical conformational transition promoted by the correct nucleotide and only preferred DNA substrates.

SUBMITTER: Nevin P

PROVIDER: S-EPMC4504746 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Noncognate DNA damage prevents the formation of the active conformation of the Y-family DNA polymerases DinB and DNA polymerase κ.

Nevin Philip P Lu Xueguang X Zhang Ke K Engen John R JR Beuning Penny J PJ

The FEBS journal 20150511 14

Y-family DNA polymerases are specialized to copy damaged DNA, and are associated with increased mutagenesis, owing to their low fidelity. It is believed that the mechanism of nucleotide selection by Y-family DNA polymerases involves conformational changes preceding nucleotidyl transfer, but there is limited experimental evidence for such structural changes. In particular, nucleotide-induced conformational changes in bacterial or eukaryotic Y-family DNA polymerases have, to date, not been extensi ...[more]

PMID: 25899385

Similar Datasets

Project description:Pseudomonas aeruginosa is a human opportunistic pathogen that chronically infects the lungs of cystic fibrosis patients and is the leading cause of morbidity and mortality of people afflicted with this disease. A striking correlation between mutagenesis and the persistence of P. aeruginosa has been reported. In other well-studied organisms, error-prone replication by Y family DNA polymerases contributes significantly to mutagenesis. Based on an analysis of the PAO1 genome sequence, P. aeruginosa contains a single Y family DNA polymerase encoded by the dinB gene. As part of an effort to understand the mechanisms of mutagenesis in P. aeruginosa, we have cloned the dinB gene of P. aeruginosa and utilized a combination of genetic and biochemical approaches to characterize the activity and regulation of the P. aeruginosa DinB protein (DinB(Pa)). Our results indicate that DinB(Pa) is a distributive DNA polymerase that lacks intrinsic proofreading activity in vitro. Modest overexpression of DinB(Pa) from a plasmid conferred a mutator phenotype in both Escherichia coli and P. aeruginosa. An examination of this mutator phenotype indicated that DinB(Pa) has a propensity to promote C-->A transversions and -1 frameshift mutations within poly(dGMP) and poly(dAMP) runs. The characterization of lexA+ and DeltalexA::aacC1 P. aeruginosa strains, together with in vitro DNA binding assays utilizing cell extracts or purified P. aeruginosa LexA protein (LexA(Pa)), indicated that the transcription of the dinB gene is regulated as part of an SOS-like response. The deletion of the dinB(Pa) gene sensitized P. aeruginosa to nitrofurazone and 4-nitroquinoline-1-oxide, consistent with a role for DinB(Pa) in translesion DNA synthesis over N2-dG adducts. Finally, P. aeruginosa exhibited a UV-inducible mutator phenotype that was independent of dinB(Pa) function and instead required polA and polC, which encode DNA polymerase I and the second DNA polymerase III enzyme, respectively. Possible roles of the P. aeruginosa dinB, polA, and polC gene products in mutagenesis are discussed.

Dataset Information

Noncognate DNA damage prevents the formation of the active conformation of the Y-family DNA polymerases DinB and DNA polymerase ?.

Publications

Noncognate DNA damage prevents the formation of the active conformation of the Y-family DNA polymerases DinB and DNA polymerase κ.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets