Variants of DNA polymerase Beta extend mispaired DNA due to increased affinity for nucleotide substrate.
DNA polymerase beta offers an attractive system to study the biochemical mechanism of polymerase-dependent mutagenesis. Variants of DNA polymerase beta, Y265F and Y265W, were analyzed for misincorporation efficiency and mispair extension ability, relative to wild-type DNA polymerase beta. Our data show that the fidelity of the mutant polymerases is similar to wild-type enzyme on a one-nucleotide gapped DNA substrate. In contrast, with a six-nucleotide gapped DNA, the mutant proteins are slightly more accurate than the wild-type enzyme. The mutagenic potential of Y265F and Y265W is more pronounced when encountering a mispaired DNA substrate. Here, both variants can extend a G:G mispair quite efficiently, and Y265F can also extend a T:G mispair. The kinetic basis of the increased mispair extension efficiency is due to an improved ability to bind to the incoming nucleotide. Y265W extends the G:G mispair even with an incorrect nucleotide substrate. Overall, our results demonstrate that the Y265 hinge residue is important for stabilizing the architecture of the nucleotide binding pocket of DNA polymerase beta, and that alterations of this residue can have significant impacts upon the fidelity of DNA synthesis.
Shah
Amit M
AM
Yale University School of Medicine, Departments of Therapeutic Radiology and Genetics, New Haven, Connecticut 06520, USA.
Maitra
Mausumi
M
Sweasy
Joann B
JB
D019943:Amino Acid Substitution; D000818:Animals; D020137:Base Pair Mismatch / Q000502:physiology*; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D007700:Kinetics; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic
D017931:DNA Primers; D003854:Deoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D019951:DNA Polymerase beta