Pre-steady-state kinetic studies of protein-template-directed nucleotide incorporation by the yeast Rev1 protein

Craig A. Howell, Satya Prakash, M. Todd Washington

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)13451-13459
Number of pages9
JournalBiochemistry
Volume46
Issue number46
DOIs
StatePublished - Nov 20 2007

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Fungal Proteins
Nucleotides
Kinetics
Proteins
DNA
DNA-Directed DNA Polymerase
Ternary Complex Factors
DNA Glycosylases
Thermodynamics
Escherichia coli
DNA Damage
Hydrogen
Hydrogen bonds
Crystal structure
X-Rays
X rays

ASJC Scopus subject areas

  • Biochemistry

Cite this

Pre-steady-state kinetic studies of protein-template-directed nucleotide incorporation by the yeast Rev1 protein. / Howell, Craig A.; Prakash, Satya; Washington, M. Todd.

In: Biochemistry, Vol. 46, No. 46, 20.11.2007, p. 13451-13459.

Research output: Contribution to journalArticle

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