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

• Published in: The Journal of biological chemistry Vol. 275; no. 19; pp. 14541 - 14549
• Main Authors: Gomes, Xavier V., Gary, Sonja L., Burgers, Peter M.J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12.05.2000; American Society for Biochemistry and Molecular Biology
• Subjects: adenosinetriphosphatase; Base Sequence; binding; cloning; Cloning, Molecular; DNA; DNA Primers; DNA Replication; DNA-binding proteins; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Escherichia coli; Escherichia coli - genetics; gene expression; Homeodomain Proteins; Humans; ligase; Ligases - genetics; Minor Histocompatibility Antigens; mutants; Mutation; phenotype; proliferating cell nuclear antigen; Protein Binding; Proto-Oncogene Proteins c-bcl-2; recombinant proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; replication factor C; Replication Protein C; Repressor Proteins; RFC gene; RFC1C gene; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins; Sequence Homology, Nucleic Acid
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.19.14541

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-ΔN, 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-ΔN 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 byrfc1-ΔN in yeast shows essentially no phenotype with regard to DNA replication, damage susceptibility, telomere length maintenance, and intrachromosomal recombination.