TY - JOUR
T1 - Mismatch repair and DNA polymerase δproofreading prevent catastrophic accumulation of leading strand errors in cells expressing a cancer-associated DNA polymerase ϵ variant
AU - Bulock, Chelsea R.
AU - Xing, Xuanxuan
AU - Shcherbakova, Polina V.
N1 - Funding Information:
National Institutes of Health [ES015869, CA239688]; Nebraska Department of Health and Human Services [LB506 to P.V.S.]; C.R.B. was supported by a University of Nebraska Medical Center Graduate Studies Research Fellowship. Funding for open access charge: National Cancer Institute [CA239688].
PY - 2020/9/18
Y1 - 2020/9/18
N2 - Substitutions in the exonuclease domain of DNA polymerase ϵ cause ultramutated human tumors. Yeast and mouse mimics of the most common variant, P286R, produce mutator effects far exceeding the effect of Polϵ exonuclease deficiency. Yeast Polϵ-P301R has increased DNA polymerase activity, which could underlie its high mutagenicity. We aimed to understand the impact of this increased activity on the strand-specific role of Polϵ in DNA replication and the action of extrinsic correction systems that remove Polϵ errors. Using mutagenesis reporters spanning a well-defined replicon, we show that both exonuclease-deficient Polϵ (Polϵ-exo-) and Polϵ-P301R generate mutations in a strictly strand-specific manner, yet Polϵ-P301R is at least ten times more mutagenic than Polϵ-exo- at each location analyzed. Thus, the cancer variant remains a dedicated leading-strand polymerase with markedly low accuracy. We further show that P301R substitution is lethal in strains lacking Polδproofreading or mismatch repair (MMR). Heterozygosity for pol2-P301R is compatible with either defect but causes strong synergistic increases in the mutation rate, indicating that Polϵ-P301R errors are corrected by Polδproofreading and MMR. These data reveal the unexpected ease with which polymerase exchange occurs in vivo, allowing Polδexonuclease to prevent catastrophic accumulation of Polϵ-P301R-generated errors on the leading strand.
AB - Substitutions in the exonuclease domain of DNA polymerase ϵ cause ultramutated human tumors. Yeast and mouse mimics of the most common variant, P286R, produce mutator effects far exceeding the effect of Polϵ exonuclease deficiency. Yeast Polϵ-P301R has increased DNA polymerase activity, which could underlie its high mutagenicity. We aimed to understand the impact of this increased activity on the strand-specific role of Polϵ in DNA replication and the action of extrinsic correction systems that remove Polϵ errors. Using mutagenesis reporters spanning a well-defined replicon, we show that both exonuclease-deficient Polϵ (Polϵ-exo-) and Polϵ-P301R generate mutations in a strictly strand-specific manner, yet Polϵ-P301R is at least ten times more mutagenic than Polϵ-exo- at each location analyzed. Thus, the cancer variant remains a dedicated leading-strand polymerase with markedly low accuracy. We further show that P301R substitution is lethal in strains lacking Polδproofreading or mismatch repair (MMR). Heterozygosity for pol2-P301R is compatible with either defect but causes strong synergistic increases in the mutation rate, indicating that Polϵ-P301R errors are corrected by Polδproofreading and MMR. These data reveal the unexpected ease with which polymerase exchange occurs in vivo, allowing Polδexonuclease to prevent catastrophic accumulation of Polϵ-P301R-generated errors on the leading strand.
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U2 - 10.1093/nar/gkaa633
DO - 10.1093/nar/gkaa633
M3 - Article
C2 - 32756902
AN - SCOPUS:85091263925
VL - 48
SP - 9124
EP - 9134
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 16
ER -