Fidelity of DNA polymerase delta holoenzyme from Saccharomyces cerevisiae: the sliding clamp proliferating cell nuclear antigen decreases its fidelity.
DNA polymerases delta and epsilon (pol delta and epsilon) are the two major replicative polymerases in the budding yeast Saccharomyces cerevisiae. The fidelity of pol delta is influenced by its 3'-5' proofreading exonuclease activity, which corrects misinsertion errors, and by enzyme cofactors. PCNA is a pol delta cofactor, called the sliding clamp, which increases the processivity of pol delta holoenzyme. This study measures the fidelity of 3'-5' exonuclease-proficient and -deficient pol delta holoenzyme using a synthetic 30mer primer/100mer template in the presence and absence of PCNA. Although PCNA increases pol delta processivity, the presence of PCNA decreased pol delta fidelity 2-7-fold. In particular, wild-type pol delta demonstrated the following nucleotide substitution efficiencies for mismatches in the absence of PCNA: G.G, 0.728 x 10(-4); T.G, 1.82 x 10(-4); A.G, <0.01 x 10(-4). In the presence of PCNA these values increased as follows: G.G, 1.30 x 10(-4); T.G, 2.62 x 10(-4); A.G, 0.074 x 10(-4). A similar but smaller effect was observed for exonuclease-deficient pol delta (i.e., 2-4-fold increase in nucleotide substitution efficiencies in the presence of PCNA). Thus, the fidelity of wild-type pol delta in the presence of PCNA is more than 2 orders of magnitude lower than the fidelity of wild-type pol epsilon holoenzyme and is comparable to the fidelity of exonuclease-deficient pol epsilon holoenzyme.
Hashimoto
Keiji
K
Laboratories for Biomolecular Networks, Graduate School of Frontier Biosciences, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
Shimizu
Kikuo
K
Nakashima
Naomi
N
Sugino
Akio
A
D004257:DNA Polymerase II / Q000737:chemistry; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043211:Exodeoxyribonuclease V / Q000737:chemistry / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013698:Templates, Genetic
D020035:Holoenzymes; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V