Replication factor C (RFC) is a five-subunit DNA polymerase accessory protein that functions as a structure-specific, DNA-dependent ATPase. The ATPase function of RFC is activated by proliferating cell nuclear antigen. RFC was originally purified from human cells on the basis of its requirement for simian virus 40 DNA replication in vitro. A functionally homologous protein complex from Saccharomyces cerevisiae, called ScRFC, has been identified. Here we report the cloning, by either peptide sequencing or by sequence similarity to the human cDNAs, of the S. cerevisiae genes RFC1, RFC2, RFC3, RFC4, and RFC5. The amino acid sequences are highly similar to the sequences of the homologous human RFC 140-, 37-, 36-, 40-, and 38-kDa subunits, respectively, and also show amino acid sequence similarity to functionally homologous proteins from Escherichia coli and the phage T4 replication apparatus. All five subunits show conserved regions characteristic of ATP/GTP-binding proteins and also have a significant degree of similarity among each other. We have identified eight segments of conserved amino acid sequences that define a family of related proteins. Despite their high degree of sequence similarity, all five RFC genes are essential for cell proliferation in S. cerevisiae. RFC1 is identical to CDC44, a gene identified as a cell division cycle gene encoding a protein involved in DNA metabolism. CDC44/RFC1 is known to interact genetically with the gene encoding proliferating cell nuclear antigen, confirming previous biochemical evidence of their functional interaction in DNA replication.

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

Models of DNA replication in yeast andMuch progress has been made identifying proteins that assemble during early stages in replication prior to the start of DNA synthesis. These events are directed by origins of replication, which orchestrate formation of the pre-replication complex (pre-RC) 1 at origins during the G 1 phase of the cell cycle. They include origin binding by the origin recognition complex and subsequent recruitment of Cdc6 and Cdt1, followed by the loading of the Mcm2-7 complex (1, 2). Further progression is regulated by the cell cycle through the activation of two S phase promoting kinases, the S phase cyclin-dependent kinase and Cdc7/Dbf4 kinase. Activation of the pre-RC by these kinases promotes Cdc45 association with origins (3, 4), events that lead subsequently to DNA unwinding and the ordered assembly of the replication fork machinery.The DNA polymerase ␣-primase (pol ␣-primase) complex also plays an essential role in initiation by synthesizing short RNA primers required to begin both leading and lagging strand DNA synthesis. The four subunit structure of pol ␣-primase is conserved, and each subunit is essential for cell viability in yeast (5, 6). The largest subunit of pol ␣-primase (p180) contains the DNA polymerase catalytic center (7) and elongates RNA primers of 8 -12 nucleotides synthesized by primase. The catalytic center of primase is located within the p48 subunit (8), which exists as a tight complex with p58 (9, 10). The p58 subunit was shown to be required for distinct aspects of primer synthesis, including initiation and elongation (11). The fourth subunit of DNA pol ␣-primase, p70/␤, is suggested to recruit the complex onto chromatin as a result of its cell cycle-regulated phosphorylated state (12, 13). Polymerase ␣-primase subunits have been reported to interact with a number of replication proteins including Cdc45 (14), Dna2 (15), RPA (16,17), and viral initiator proteins (16,18,19), consistent with the idea that replication forks are large precisely assembled multiprotein complexes.Mcm10p has been implicated both in the initiation and elongation steps of DNA replication. MCM10 was first identified in Saccharomyces cerevisiae using screens for mutants defective in DNA replication and for the stable maintenance of plasmids (20,21). Initiation at replication origins is drastically reduced in the mcm10-1 mutant, and replication across origins is impeded (21,22). Mutations in MCM10 result in a delay in the completion of DNA synthesis after cells are released from HU arrest (23), suggesting that Mcm10p is essential for continued fork progression. MCM10 mutants are suppressed by mutant MCM5 and MCM7 genes and are synthetically lethal with mutant genes of ORC, CDC45, DNA2, DPB11, and genes encoding subunits of DNA polymerase ␦ and ⑀ (22-27). Seven new mutants named slm1-slm6 for synthetically lethal with mcm10 include mutations in genes that are allelic to MCM7, MCM2, CDC45, DNA2, and mutations in novel DNA repair genes represented by SLM2 and SLM6 (28). Biochemical and genetic int...

DNA replication occurs through a complex series of reactions that are mechanistically coordinated at the replication fork. Studies of model DNA replication systems indicate that origin recognition by an initiator protein permits assembly and activation of the replicative helicase at the origin. Helicases unwind duplex DNA to generate a single-strand template on which primases can initiate synthesis of RNA primers that are 4 -20 nucleotides in length and that are extended by DNA polymerases to make Okazaki fragments (1). Primases are physically coupled to the replicative DNA helicases and DNA polymerases, which translocate together through the duplex, resulting in DNA unwinding coincident with synthesis of RNA primers and their extension with deoxynucleotides.In eukaryotes, the Mcm proteins are essential replication factors that were identified as proteins required for minichromosomal maintenance in a genetic screen for mutants defective in initiation of replication (2). Six of these members are sequencerelated proteins, Mcm2-7, which interact to form a hexameric complex. Although a substantial body of data suggests that the Mcm2-7p complex acts as the replicative helicase (3), helicase activity has been detected only in the Mcm4-6-7 subcomplex (4 -6). Prior to the initiation of replication, the Mcm2-7 complex associates with the initiator at replication origins. At the G 1 /S transition, both S phase cyclin-dependent kinase and Cdc7p-Dbf4p kinase activities rise, promoting the maturation of the pre-replicative complex to the preinitiation complex (7). Phosphorylation of the chromatin-bound Mcm2-7 complex by the Cdc7-Dbf4 kinase (8) Previously, we reported that, in vitro, S. pombe Mcm10p (amino acids 1-593; SpMcm10p) binds preferentially to singlestranded DNA (ssDNA) and to the large subunit of the pol ␣-primase complex and activates its DNA synthetic activity (25). In addition, we found that Mcm10p facilitates the binding of the pol ␣-primase complex to primed DNA and forms a stable ternary complex, suggesting that Mcm10p recruits the pol ␣-primase complex to template DNA. In keeping with these in vitro findings, Mcm10p was shown recently to stabilize the large subunit of the pol ␣-primase complex in S. cerevisiae (15) as well as the chromatin association of the pol ␣-primase complex in S. pombe (26).In this study, we demonstrate that full-length SpMcm10p (amino acids 1-593) and its C-terminal fragment (amino acids 416 -593) contain primase activity that catalyzes the synthesis of oligoribonucleotides that are extended by Escherichia coli pol I. Primase activity both co-sedimented and co-eluted with these full-length and truncated proteins, although their hydro-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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.

No abstract

No abstract

No abstract