Unique Error Signature of the Four-subunit Yeast DNA Polymerase ε

Polina V Shcherbakova, Youri I Pavlov, Olga Chilkova, Igor B. Rogozin, Erik Johansson, Thomas A. Kunkel

Research output: Contribution to journalArticle

85 Citations (Scopus)

Abstract

We have purified wild type and exonuclease-deficient four-subunit DNA polymerase ε (Pol ε) complex from Saccharomyces cerevisiae and analyzed the fidelity of DNA synthesis by the two enzymes. Wild type Pol ε synthesizes DNA accurately, generating single-base substitutions and deletions at average error rates of ≤2 × 10-5 and ≤5 × 10-7, respectively. Pol ε lacking 3′ → 5′ exonuclease activity is less accurate to a degree suggesting that wild type Pol ε proofreads at least 92% of base substitution errors and at least 99% of frameshift errors made by the polymerase. Surprisingly the base substitution fidelity of exonuclease-deficient Pol ε is severalfold lower than that of proofreading-deficient forms of other replicative polymerases. Moreover the spectrum of errors shows a feature not seen with other A, B, C, or X family polymerases: a high proportion of transversions resulting from T-dTTP, T-dCTP, and C-dTTP mispairs. This unique error specificity and amino acid sequence alignments suggest that the structure of the polymerase active site of Pol ε differs from those of other B family members. We observed both similarities and differences between the spectrum of substitutions generated by proofreading-deficient Pol ε in vitro and substitutions occurring in vivo in a yeast strain defective in Pol ε proofreading and DNA mismatch repair. We discuss the implications of these findings for the role of Pol ε polymerase activity in DNA replication.

Original languageEnglish (US)
Pages (from-to)43770-43780
Number of pages11
JournalJournal of Biological Chemistry
Volume278
Issue number44
DOIs
StatePublished - Oct 31 2003

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Exonucleases
DNA-Directed DNA Polymerase
Yeast
Substitution reactions
Yeasts
DNA Mismatch Repair
Sequence Alignment
DNA Replication
Saccharomyces cerevisiae
Amino Acid Sequence
Catalytic Domain
DNA
Enzymes
Repair
Amino Acids
thymidine 5'-triphosphate

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Unique Error Signature of the Four-subunit Yeast DNA Polymerase ε. / Shcherbakova, Polina V; Pavlov, Youri I; Chilkova, Olga; Rogozin, Igor B.; Johansson, Erik; Kunkel, Thomas A.

In: Journal of Biological Chemistry, Vol. 278, No. 44, 31.10.2003, p. 43770-43780.

Research output: Contribution to journalArticle

Shcherbakova, Polina V ; Pavlov, Youri I ; Chilkova, Olga ; Rogozin, Igor B. ; Johansson, Erik ; Kunkel, Thomas A. / Unique Error Signature of the Four-subunit Yeast DNA Polymerase ε. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 44. pp. 43770-43780.
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