Identification of Replication Factor C from Saccharomyces cerevisiae: A Component of the Leading-Strand DNA Replication Complex

Abstract

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.

Characterization of the DNA-dependent ATPase and a DNA unwinding activity associated with the yeast DNA polymerase .alpha. complex

“…With both poly(dA) and poly(dT) DNA, increasing the primentemplate ratio did not stimulate the activity, and the activity was actually diminished with an increase in the ratio which is likely due to the conversion of ssDNA to dsDNA. Therefore, this ATPase was purely ssDNA-dependent and did not have any preference for the primer-template junction like that reported for RF-C (Fien & Stillman, 1992).…”

“…In addition, several DNA-dependent eukaryotic ATPases have been reported recently (Vishwanath & Baril, 1990;Tsurimoto & Stillman, 1989;Lee etal., 1991;Fien & Stillman, 1992). Replication factor C (RF-C) is known to be a stimulator of DNA polymerase 5, functions in the replication of the leading strand (Lee et al, 1991), and is stimulated by the 3'-OH termini of primers in a primed DNA template.…”

DNA polymerase .epsilon. interacts with proliferating cell nuclear antigen in primer recognition and elongation

“…One unit of enzyme activity corresponds to the incorporation of 1 nmol of total dTMP into acid-precipitable material in 60 min at 37 °C in a standard assay containing 0.5 pg (nucleotides) of poly(dA)/ oligo(dT)io:i and 20 /iM dTTP. Human PCNA was overexpressed in E. coli strain BL21(DE3) harboring the expression plasmid pT7/PCNA and purified as described by Fien and Stillman (1992).…”

Phosphorylation of the PCNA Binding Domain of the Large Subunit of Replication Factor C by Ca2+/Calmodulin-Dependent Protein Kinase II Inhibits DNA Synthesis

“…Recombinant GSTfused CaMKI expressed in E. coli and recombinant R-CaMKII and CaMKIV expressed in baculovirus were a gift of A. R. Means . Human PCNA was overexpressed in E. coli strain BL21(DE3) harboring the expression plasmid pT7/PCNA and purified as described by Fien and Stillman (1992). Recombinant, phosphorylatable human PCNA was prepared as described (Podust et al, 1995).…”