Mechanism of efficient and accurate nucleotide incorporation opposite 7,8-dihydro-8-oxoguanine by Saccharomyces cerevisiae DNA polymerase eta.
Most DNA polymerases incorporate nucleotides opposite template 7,8-dihydro-8-oxoguanine (8-oxoG) lesions with reduced efficiency and accuracy. DNA polymerase (Pol) eta, which catalyzes the error-free replication of template thymine-thymine (TT) dimers, has the unique ability to accurately and efficiently incorporate nucleotides opposite 8-oxoG templates. Here we have used pre-steady-state kinetics to examine the mechanisms of correct and incorrect nucleotide incorporation opposite G and 8-oxoG by Saccharomyces cerevisiae Pol eta. We found that Pol eta binds the incoming correct dCTP opposite both G and 8-oxoG with similar affinities, and it incorporates the correct nucleotide bound opposite both G and 8-oxoG with similar rates. While Pol eta incorporates an incorrect A opposite 8-oxoG with lower efficiency than it incorporates a correct C, it does incorporate A more efficiently opposite 8-oxoG than opposite G. This is mainly due to greater binding affinity for the incorrect incoming dATP opposite 8-oxoG. Overall, these results show that Pol eta replicates through 8-oxoG without any barriers introduced by the presence of the lesion.
Carlson
Karissa D
KD
Department of Biochemistry, 4-403 Bowen Science Building, University of Iowa, Iowa City, IA 52242-1109, USA.
Washington
M Todd
MT
D020029:Base Pairing; D004249:DNA Damage / Q000235:genetics; D004261:DNA Replication / Q000235:genetics / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic; D000099270:Y-Family DNA Polymerases
D004259:DNA-Directed DNA Polymerase; D006147:Guanine; D009711:Nucleotides; D029701:Saccharomyces cerevisiae Proteins; C453560:7,8-dihydro-8-oxoguanine; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases
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