Characterization of a replicative DNA polymerase mutant with reduced fidelity and increased translesion synthesis capacity
Open Access
- 23 May 2008
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 36 (12), 3892-3904
- https://doi.org/10.1093/nar/gkn312
Abstract
Changing a highly conserved amino acid in motif A of any of the four yeast family B DNA polymerases, DNA polymerase α, δ, ɛ or ζ, results in yeast strains with elevated mutation rates. In order to better understand this phenotype, we have performed structure–function studies of homologous mutants of RB69 DNA polymerase (RB69 pol), a structural model for family B members. When Leu415 in RB69 pol is replaced with phenylalanine or glycine, the mutant polymerases retain high-catalytic efficiency for correct nucleotide incorporation, yet have increased error rates due to increased misinsertion, increased mismatch extension and inefficient proofreading. The Leu415Phe mutant also has increased dNTP insertion efficiency opposite a template 8-oxoG and opposite an abasic site. The 2.5 Å crystal structure of a ternary complex of RB69 L415F pol with a correctly base-paired incoming dTTP reveals that the phenylalanine ring is accommodated within a cavity seen in the wild-type enzyme, without steric clash or major change in active site geometry, consistent with retention of high-catalytic efficiency for correct incorporation. In addition, slight structural differences were observed that could be relevant to the reduced fidelity of L415F RB69 pol.Keywords
This publication has 33 references indexed in Scilit:
- Division of Labor at the Eukaryotic Replication ForkMolecular Cell, 2008
- Mutator alleles of yeast DNA polymerase ζDNA Repair, 2007
- Mutation at the Polymerase Active Site of Mouse DNA Polymerase δ Increases Genomic Instability and Accelerates TumorigenesisMolecular and Cellular Biology, 2007
- Yeast DNA Polymerase ε Participates in Leading-Strand DNA ReplicationScience, 2007
- Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymeraseNucleic Acids Research, 2007
- Inefficient Proofreading and Biased Error Rates during Inaccurate DNA Synthesis by a Mutant Derivative of Saccharomyces cerevisiae DNA Polymerase δJournal of Biological Chemistry, 2007
- RPA and PCNA suppress formation of large deletion errors by yeast DNA polymerase δNucleic Acids Research, 2006
- Evidence that Errors Made by DNA Polymerase α are Corrected by DNA Polymerase δCurrent Biology, 2006
- Coot: model-building tools for molecular graphicsActa Crystallographica Section D-Biological Crystallography, 2004
- Structure of the Replicating Complex of a Pol α Family DNA PolymeraseCell, 2001