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Papers In Press, published online ahead of print December 26, 2001
J. Biol. Chem, 10.1074/jbc.M108546200
Submitted on September 6, 2001
Revised on December 12, 2001
Accepted on December 25, 2001
in insect cells: Purification and characterization
Divisions of Biology and Chemistry, California Institute of Technology, Pasadena, CA 91024
Corresponding Author: jcampbel{at}its.caltech.edu
DNA polymerase epsilon (pol 
) is a multiple subunit complex consisting of at least four proteins, including catalytic Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol has been shown to play essential roles in chromosomal DNA replication. Here, we report reconstitution of the yeast pol complex, which was expressed and purified from baculovirus-infected insect cells. During the purification, we were able to resolve the pol complex and truncated 140 kDa Pol2p, as was initially observed with the pol purified from yeast. Biochemical characterization of subunit stoichiometry, salt sensitivity, processivity and stimulation by PCNA indicates that the reconstituted pol is functionally identical to native pol purified from yeast, and therefore useful for biochemical characterization of the interactions of pol with other replication, recombination and repair proteins. Identification and characterization of a PCNA consensus interaction domain on Pol2p indicates that the motif is dispensable for DNA replication but is important for MMS 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.
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