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J. Biol. Chem., Vol. 278, Issue 44, 43770-43780, October 31, 2003

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Unique Error Signature of the Four-subunit Yeast DNA Polymerase ^*

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

From the Laboratory of Molecular Genetics and the Laboratory of Structural Biology, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, the Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden, and the ^||National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20894

We have purified wild type and exonuclease-deficient four-subunit DNA polymerase (Pol ) complex from Saccharomyces cerevisiae and analyzed the fidelity of DNA synthesis by the two enzymes. Wild type Pol synthesizes DNA accurately, generating single-base substitutions and deletions at average error rates of 2 x 10^-5 and 5 x 10^-7, respectively. Pol lacking 3' 5' exonuclease activity is less accurate to a degree suggesting that wild type Pol 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 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 differs from those of other B family members. We observed both similarities and differences between the spectrum of substitutions generated by proofreading-deficient Pol in vitro and substitutions occurring in vivo in a yeast strain defective in Pol proofreading and DNA mismatch repair. We discuss the implications of these findings for the role of Pol polymerase activity in DNA replication.

Received for publication, June 27, 2003 , and in revised form, July 25, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by the Swedish Research Council, the Swedish Cancer Society, the Magnus Bergwalls stiftelse, the Åke Wibergs Stiftelse, and the Medical Faculty of Umeå University.

^** Supported in part by Russian Fund for Basic Research Grant N 02-04-48342. Permanent address: Inst. of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk, 630090 Russia.

To whom correspondence should be addressed: Laboratory of Structural Biology, NIEHS, National Institutes of Health, Dept. of Health and Human Services, Research Triangle Park, NC 27709. Tel.: 919-541-2644; Fax: 919-541-7613; E-mail: kunkel{at}niehs.nih.gov.

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