TY - JOUR
T1 - Inefficient proofreading and biased error rates during inaccurate DNA synthesis by a mutant derivative of Saccharomyces cerevisiae DNA polymerase
AU - McElhinny, Stephanie A.Nick
AU - Stith, Carrie M.
AU - Burgers, Peter M.J.
AU - Kunkel, Thomas A.
PY - 2007/1/26
Y1 - 2007/1/26
N2 - DNA polymerase δ (pol δ) is a high fidelity eukaryotic enzyme that participates in DNA repair and is essential for DNA replication. Toward the goal of dissecting its multiple biological functions, here we describe the biochemical properties of Saccharomyces cerevisiae pol δ with a methionine replacing conserved leucine 612 at the polymerase active site. Compared with wild type pol δ, L612M pol δhas normal processivity and slightly higher polymerase specific activity. L612M pol δ also has normal 3′ exonuclease activity, yet it is impaired in partitioning mismatches to the exonuclease active site, thereby reducing DNA synthesis fidelity. Error rates in vitro for L612M pol δ are elevated for both base substitutions and single base deletions but in a highly biased manner. For each of the six possible pairs of reciprocal mismatches that could arise during replication of complementary DNA strands to account for any particular base substitution in vivo (e.g. T-dGMP or A-dCMP for T to C transitions), L612M pol δ error rates are substantially higher for one mismatch than the other. These results provide a biochemical explanation for our observation, which confirms earlier genetic studies, that a haploid pol3-L612M S. cerevisiae strain has an elevated spontaneous mutation rate that is likely due to reduced replication fidelity in vivo.
AB - DNA polymerase δ (pol δ) is a high fidelity eukaryotic enzyme that participates in DNA repair and is essential for DNA replication. Toward the goal of dissecting its multiple biological functions, here we describe the biochemical properties of Saccharomyces cerevisiae pol δ with a methionine replacing conserved leucine 612 at the polymerase active site. Compared with wild type pol δ, L612M pol δhas normal processivity and slightly higher polymerase specific activity. L612M pol δ also has normal 3′ exonuclease activity, yet it is impaired in partitioning mismatches to the exonuclease active site, thereby reducing DNA synthesis fidelity. Error rates in vitro for L612M pol δ are elevated for both base substitutions and single base deletions but in a highly biased manner. For each of the six possible pairs of reciprocal mismatches that could arise during replication of complementary DNA strands to account for any particular base substitution in vivo (e.g. T-dGMP or A-dCMP for T to C transitions), L612M pol δ error rates are substantially higher for one mismatch than the other. These results provide a biochemical explanation for our observation, which confirms earlier genetic studies, that a haploid pol3-L612M S. cerevisiae strain has an elevated spontaneous mutation rate that is likely due to reduced replication fidelity in vivo.
UR - http://www.scopus.com/inward/record.url?scp=34047260752&partnerID=8YFLogxK
U2 - 10.1074/jbc.M609591200
DO - 10.1074/jbc.M609591200
M3 - Article
C2 - 17121822
AN - SCOPUS:34047260752
VL - 282
SP - 2324
EP - 2332
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 4
ER -