Probing the mechanisms of two exonuclease domain mutators of DNA polymerase ϵ.
Joseph M Dahl, Natalie Thomas, Maxwell A Tracy, Brady L Hearn, Lalith Perera, Scott R Kennedy, Alan J Herr, Thomas A Kunkel
Author Information
Joseph M Dahl: Genome Integrity Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, Research Triangle Park, NC 27709, USA.
Natalie Thomas: Genome Integrity Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, Research Triangle Park, NC 27709, USA.
Maxwell A Tracy: Department of Laboratory Medicine and Pathology, UW Medicine, Seattle, WA 98195, USA.
Brady L Hearn: Department of Laboratory Medicine and Pathology, UW Medicine, Seattle, WA 98195, USA.
Lalith Perera: Genome Integrity Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, Research Triangle Park, NC 27709, USA.
Scott R Kennedy: Department of Laboratory Medicine and Pathology, UW Medicine, Seattle, WA 98195, USA. ORCID
Alan J Herr: Department of Laboratory Medicine and Pathology, UW Medicine, Seattle, WA 98195, USA.
Thomas A Kunkel: Genome Integrity Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, Research Triangle Park, NC 27709, USA. ORCID
We report the properties of two mutations in the exonuclease domain of the Saccharomyces cerevisiae DNA polymerase ϵ. One, pol2-Y473F, increases the mutation rate by about 20-fold, similar to the catalytically dead pol2-D290A/E290A mutant. The other, pol2-N378K, is a stronger mutator. Both retain the ability to excise a nucleotide from double-stranded DNA, but with impaired activity. pol2-Y473F degrades DNA poorly, while pol2-N378K degrades single-stranded DNA at an elevated rate relative to double-stranded DNA. These data suggest that pol2-Y473F reduces the capacity of the enzyme to perform catalysis in the exonuclease active site, while pol2-N378K impairs partitioning to the exonuclease active site. Relative to wild-type Pol ϵ, both variants decrease the dNTP concentration required to elicit a switch between proofreading and polymerization by more than an order of magnitude. While neither mutation appears to alter the sequence specificity of polymerization, the N378K mutation stimulates polymerase activity, increasing the probability of incorporation and extension of a mismatch. Considered together, these data indicate that impairing the primer strand transfer pathway required for proofreading increases the probability of common mutations by Pol ϵ, elucidating the association of homologous mutations in human DNA polymerase ϵ with cancer.
