Pre-steady-state kinetic studies of protein-template-directed nucleotide incorporation by the yeast Rev1 protein.
The yeast Rev1 protein (Rev1p) is a member of the Y family of DNA polymerases that specifically catalyzes the incorporation of C opposite template G and several types of DNA damage. The X-ray crystal structure of the Rev1p-DNA-dCTP ternary complex showed that Rev1p utilizes an unusual mechanism of nucleotide incorporation whereby the template residue is displaced from the DNA double helix and the side chain of Arg-324 forms hydrogen bonds with the incoming dCTP. To better understand the impact of this protein-template-directed mechanism on the thermodynamics and kinetics of nucleotide incorporation, we have carried out pre-steady-state kinetic studies with Rev1p. Interestingly, we found that Rev1p's specificity for incorporating C is achieved solely at the initial nucleotide-binding step, not at the subsequent nucleotide-incorporation step. In this respect, Rev1p differs from all previously investigated DNA polymerases. We also found that the base occupying the template position in the DNA impacts nucleotide incorporation more at the nucleotide-binding step than at the nucleotide-incorporation step. These studies provide the first detailed, quantitative information regarding the mechanistic impact of protein-template-directed nucleotide incorporation by Rev1p. Moreover, on the basis of these findings and on structures of the unrelated Escherichia coli MutM DNA glycosylase, we suggest the possible structures for the ternary complexes of Rev1p with the other incoming dNTPs.
Howell
Craig A
CA
Department of Biochemistry, University of Iowa College of Medicine, Iowa City, Iowa 52242-1109, USA.
Prakash
Satya
S
Washington
M Todd
MT
D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013379:Substrate Specificity; D000099270:Y-Family DNA Polymerases
D004271:DNA, Fungal; D003854:Deoxyribonucleotides; D009713:Nucleotidyltransferases; D029701:Saccharomyces cerevisiae Proteins; C100951:REV1 protein, S cerevisiae; D000099270:Y-Family DNA Polymerases