Overproduction in Escherichia coli and characterization of yeast replication factor C lacking the ligase homology domain.
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-DeltaN, 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-DeltaN 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-DeltaN in yeast shows essentially no phenotype with regard to DNA replication, damage susceptibility, telomere length maintenance, and intrachromosomal recombination.
Gomes
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Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Gary
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SL
Burgers
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PM
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D018398:Homeodomain Proteins; D006801:Humans; D008025:Ligases / Q000235:genetics*; D015778:Minor Histocompatibility Antigens; D009154:Mutation; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D051818:Replication Protein C; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012689:Sequence Homology, Nucleic Acid
C096816:BCL2-related protein A1; D017931:DNA Primers; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D051818:Replication Protein C; D008025:Ligases