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Originally published In Press as doi:10.1074/jbc.M108546200 on December 26, 2001

J. Biol. Chem., Vol. 277, Issue 10, 7889-7896, March 8, 2002
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In Vivo Reconstitution of Saccharomyces cerevisiae DNA Polymerase epsilon  in Insect Cells
PURIFICATION AND CHARACTERIZATION*

Rajiv DuaDagger , Daniel L. Levy§, Caroline M. Li, Peter M. Snow, and Judith L. Campbell

From the Braun Laboratories, California Institute of Technology, Pasadena, California 91125

DNA polymerase epsilon  (pol epsilon ) is a multiple subunit complex consisting of at least four proteins, including catalytic Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol epsilon  has been shown to play essential roles in chromosomal DNA replication. Here, we report reconstitution of the yeast pol epsilon  complex, which was expressed and purified from baculovirus-infected insect cells. During the purification, we were able to resolve the pol epsilon  complex and truncated Pol2p (140 kDa), as was observed initially with the pol epsilon  purified from yeast. Biochemical characterization of subunit stoichiometry, salt sensitivity, processivity, and stimulation by proliferating cell nuclear antigen indicates that the reconstituted pol epsilon  is functionally identical to native pol epsilon  purified from yeast and is therefore useful for biochemical characterization of the interactions of pol epsilon  with other replication, recombination, and repair proteins. Identification and characterization of a proliferating cell nuclear antigen consensus interaction domain on Pol2p indicates that the motif is dispensable for DNA replication but is important for methyl methanesulfonate damage-induced DNA repair. Analysis of the putative zinc finger domain of Pol2p for zinc binding capacity demonstrates that it binds zinc. Mutations of the conserved cysteines in the putative zinc finger domain reduced zinc binding, indicating that cysteine ligands are directly involved in binding zinc.

* This work was supported by Public Health Service Grants GM25508 and F32 GM63374-01.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Hyseq Inc., 670 Almanor Ave., Sunnyvale, CA 94086.

§ Present address: Dept. of Biochemistry and Biophysics, Box 0448, University of California, San Francisco, CA 94143.

To whom correspondence should be addressed: Braun Laboratories 147-75, California Institute of Technology, 1200 East California Blvd., Pasadena, CA 91125. Tel.: 626-395-6053; Fax: 626-405-9452; E-mail: jcampbel@cco.caltech.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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