Incorrect nucleotide insertion at the active site of a G:A mismatch catalyzed by DNA polymerase β

doi:10.1073/pnas.0801257105

Incorrect nucleotide insertion at the active site of a G:A mismatch catalyzed by DNA polymerase β

^†Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599; and
^‡Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709-2233

Communicated by Robert G. Parr, University of North Carolina, Chapel Hill, NC, February 7, 2008 (received for review August 18, 2007)

Abstract

Based on a recent ternary complex crystal structure of human DNA polymerase β with a G:A mismatch in the active site, we carried out a theoretical investigation of the catalytic mechanism of incorrect nucleotide incorporation using molecular dynamics simulation, quantum mechanics, combined quantum mechanics, and molecular mechanics methods. A two-stage mechanism is proposed with a nonreactive active-site structural rearrangement prechemistry step occurring before the nucleotidyl transfer reaction. The free energy required for formation of the prechemistry state is found to be the major factor contributing to the decrease in the rate of incorrect nucleotide incorporation compared with correct insertion and therefore to fidelity enhancement. Hence, the transition state and reaction barrier for phosphodiester bond formation after the prechemistry state are similar to that for correct insertion reaction. Key residues that provide electrostatic stabilization of the transition state are identified.

Footnotes

^§To whom correspondence should be addressed. E-mail: lee_pedersen{at}unc.edu
Author contributions: P.L., V.K.B., L.C.P., W.A.B., S.H.W., and L.G.P. designed research, performed research, contributed new reagents/analytic tools, analyzed data, and wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0801257105/DCSupplemental.