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The Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase ␦ holoenzyme activity was achieved at 37 C but not at 14 C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase ␦ but completely defective for interaction with DNA polymerase . Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.

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-⌬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-⌬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-⌬N in yeast shows essentially no phenotype with regard to DNA replication, damage susceptibility, telomere length maintenance, and intrachromosomal recombination.DNA replication in eukaryotes is a complex process involving a large number of proteins. In yeast, processive DNA synthesis is performed by DNA polymerase ␦ (Pol ␦) 1 and DNA polymerase ⑀. Two accessory factors are also required for processivity, the proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C) (1, 2). Yeast PCNA is a ring-shaped homotrimer with a monomer mass of 29 kDa (3). It functions by encircling the DNA and interacting with Pol ␦ or Pol ⑀ to maintain highly processive DNA replication (4 -7). PCNA also interacts with several other replication and repair proteins, including the replication inhibitor p21, the flap-specific endonuclease FEN-1, and DNA ligase 1 (reviewed in Ref. 8).RF-C is a multisubunit complex that binds preferentially to the 3Ј-end of template-primer junctions and is essential for loading PCNA onto DNA (9). RF-C has an associated singlestranded DNA-dependent ATPase activity that is stimulated by the presence of primer termini and PCNA (9 -11). The mechanism by which RF-C loads PCNA onto DNA is not well understood. RF-C may recognize and bind to template-primer junctions and subsequently load PCNA in an ATP-dependent manner (12). Alternatively, RF-C may form an ATP-dependent complex with PCNA, open the trimeric ring, and, upon binding a template-primer junction, close the ring around the DNA with hydrolysis of ATP (13).Yeast RF-C consists of a large subunit wit...

Yeast replication factor C (RF-C) is a heteropentamer encoded by the RFC1-5 genes. RF-C activity in yeast extracts was overproduced about 80-fold after induction of a strain containing all five genes on a single plasmid, with expression of each gene placed under control of the galactose-inducible GAL1-10 promoter. This strongly indicates that overexpression of the five known RFC genes is sufficient for overproduction of RF-C. Overexpression of all five genes was also necessary to achieve overproduction of RF-C as omission of any single gene from the plasmid gave uninduced, i.e. normal cellular levels of RF-C. The interaction between RF-C and proliferating cell nuclear antigen (PCNA) was studied with PCNAagarose beads. Binding of RF-C to PCNA-agarose beads is negligible in buffers containing 0.3 M NaCl. However, addition of Mg-ATP to the binding buffer caused strong binding of RF-C to the beads even at 0.8 M NaCl. Binding of ATP, but not its hydrolysis, was required for the strong binding mode as nonhydrolyzable analogs were also effective. The existence of two distinct binding modes between PCNA and RF-C was used as the key step in a greatly improved procedure for the purification of RF-C. RF-C from the overproduction strain purified by this procedure was essentially homogeneous and had a severalfold higher specific activity than RF-C preparations that had previously been purified through multicolumn procedures.

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