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J Biol Chem, Vol. 275, Issue 19, 14541-14549, May 12, 2000

Overproduction in Escherichia coli and Characterization of Yeast Replication Factor C Lacking the Ligase Homology Domain*

Xavier V. GomesDagger , Sonja L. Gary, and Peter M. J. Burgers§

From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

Eukaryotic replication factor C (RF-C) is a heteropentameric complex that is required to load the replication clamp proliferating cell nuclear antigen onto primed DNA. Saccharomyces cerevisiae RF-C is encoded by the genes RFC1-RFC5. The RFC1 gene was cloned under control of the strong inducible bacteriophage T7 promoter, yet induction did not yield detectable Rfc1p. However, a truncated form of RFC1 deleted for the coding region for amino acids 3-273, rfc1-Delta N, did allow overproduction. The other four RFC genes were cloned into the latter plasmid to yield a single plasmid that overproduced RF-C to moderate levels. Overproduction of the complex was further enhanced when the Escherichia coli argU gene encoding the rare arginine tRNA was also overproduced. The enzyme thus produced in E. coli was purified to homogeneity through three column steps, including a proliferating cell nuclear antigen affinity column. This enzyme, as well as the enzyme purified from yeast, is prone to aggregation and inactivation, and therefore, light scattering was used to determine conditions stabilizing the enzyme and preventing aggregation. Broad-range carrier ampholytes at about 0.05% were found to be most effective. In some assays, the Rfc1-Delta N containing RF-C from E. coli showed an increased activity compared with the full-length enzyme from yeast, likely because the latter enzyme exhibits significant nonspecific binding to single-stranded DNA. Replacement of RFC1 by rfc1-Delta N in yeast shows essentially no phenotype with regard to DNA replication, damage susceptibility, telomere length maintenance, and intrachromosomal recombination.

* This work was supported in part by Grant GM32431 from the National Institutes of Health.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 Supported in part by a fellowship from the W. M. Keck Foundation.

§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid, St. Louis, MO 63110. Tel.: 314-362-3872; Fax: 314-362-7183; E-mail: burgers@biochem.wustl.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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