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 8 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 8 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 8 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.The study of eukaryotic DNA replication has been dependent, in part, on the isolation of enzymes involved in DNA synthesis. In the absence of well-characterized mammalian origins of DNA replication, the mammalian DNA viruses, particularly simian virus 40 (SV40), have served as good model systems for understanding some aspects of eukaryotic DNA replication. The SV40 system has been used to identify proteins from human cells that are required for DNA replication from the virus origin sequences. While origins of DNA replication have been characterized in Saccharomyces cerevisiae, a primary hindrance in the study of yeast DNA replication has been the inability to reconstitute initiation of DNA replication with cellular extracts.The SV40 DNA replication system is based on the observation that protein extracts from primate cells, when supplemented with purified SV40 large T antigen (TAg), efficiently replicate plasmid DNAs containing the SV40 origin of DNA replication (24,25,41,52

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

“…M13mp2opl4 DNA was propagated in E. coli JM109 cells as described by Sanderson and Mosbaugh [30], and single-stranded M13mp2op14 DNA was purified from the culture supernatant using the CTAB DNA precipitation method [31,32]. The single-stranded M13 DNA circles were annealed to a complementary 5′-phosphorylated oligonucleotide (5′-CCCAGTCACGTCATTGTAAAACG-3′) in reactions (6 μ1 × 50 μ1) containing 20 mM Tris-HCl pH 7.4, 2 mM MgCl 2 , 50 mM NaCl, 0.4 μM ssM13, and 1.2 μM 5′-end phosphorylated oligonucleotide.…”

“…PCNA LC59 was overproduced in E. coli BL21 (DE3) and purified as described previously for wild-type PCNA, except that the LC59 double mutant flowed through, rather than binding to, the phenyl-Sepharose column [32].…”

“…When subjected to Sephacryl S-300 size exclusion chromatography, the purified his 6 -tagged PCNA preparation eluted as a −85 kDa homotrimer, indicative of wild-type functionality (Section 3.4). The PCNA double mutant, LC59A, was produced without an affinity tag and purified using the method of Fien and Stillman [32] with one modification. While wild-type PCNA bound to phenyl-Sepharose at 1.2 M NaCl, PCNA LC59 flowed through.…”

The DNA Repair Endonuclease XPG Binds to Proliferating Cell Nuclear Antigen (PCNA) and Shares Sequence Elements with the PCNA-binding Regions of FEN-1 and Cyclin-dependent Kinase Inhibitor p21

“…Human PCNA was purified from bacterial lysate by QSepharose, S-300 Sephacryl gel filtration, and Phenyl-Superose chromatography (19). Protein concentrations were determined by a Bradford-based assay (Bio-Rad) and verified by Coomassie Blue staining of SDS-PAGE gels.…”

DNA polymerase ε encoded by cdc20+ is required for chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe

“…In addition, it has been shown that two additional polypeptides are copurified with the activity of DNA polymerase III (Hashimoto et al 1998). Together with DNA polymerase II or III, three accessory protein complexes, RF-A (RP-A), RF-C, and PCNA, are required for a highly processive DNA synthesis in vitro (Burgers 1991;Yoder & Burgers 1991;Fien & Stillman 1992).…”