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Molecular and Cellular Biology, March 2005, p. 2169-2176, Vol. 25, No. 6
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.6.2169-2176.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Mechanism of Efficient and Accurate Nucleotide Incorporation Opposite 7,8-Dihydro-8-Oxoguanine by Saccharomyces cerevisiae DNA Polymerase {eta}

Karissa D. Carlson1 and M. Todd Washington1*

Department of Biochemistry, University of Iowa College of Medicine, Iowa City, Iowa1

Received 12 November 2004/ Accepted 8 December 2004

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.

* Corresponding author. Mailing address: Department of Biochemistry, 4-403 Bowen Science Building, University of Iowa, Iowa City, IA 52242-1109. Phone: (319) 335-7518. Fax: (319) 335-9570. E-mail: todd-washington{at}uiowa.edu.

Molecular and Cellular Biology, March 2005, p. 2169-2176, Vol. 25, No. 6
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.6.2169-2176.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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