The RFC2 gene encoding a subunit of replication factor C of Saccharomyces cerevisiae.

Replication Factor C (RF-C) of Saccharomyces cerevisiae is a complex that consists of several different polypeptides ranging from 120- to 37 kDa (Yoder and Burgers, 1991; Fien and Stillman, 1992), similar to human RF-C. We have isolated a gene, RFC2, that appears to be a component of the yeast RF-C. The RFC2 gene is located on chromosome X of S. cerevisiae and is essential for cell growth. Disruption of the RFC2 gene led to a dumbbell-shaped terminal morphology, common to mutants having a defect in chromosomal DNA replication. The steady-state levels of RFC2 mRNA fluctuated less during the cell cycle than other genes involved in DNA replication. Nucleotide sequence of the gene revealed an open reading frame corresponding to a polypeptide with a calculated Mr of 39,716 and a high degree of amino acid sequence homology to the 37-kDa subunit of human RF-C. Polyclonal antibodies against bacterially expressed Rfc2 protein specifically reduced RF-C activity in the RF-C-dependent reaction catalyzed by yeast DNA polymerase III. Furthermore, the Rfc2 protein was copurified with RF-C activity throughout RF-C purification. These results strongly suggest that the RFC2 gene product is a component of yeast RF-C. The bacterially expressed Rfc2 protein preferentially bound to primed single-strand DNA and weakly to ATP.     

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.     

The large subunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-like protease during Fas-mediated apoptosis.

Caspase-3 is an ICE-like protease activated during apoptosis induced by different stimuli. Poly(ADP-ribose) polymerase (PARP), the first characterized substrate of caspase-3, shares a region of homology with the large subunit of Replication Factor C (RF-C), a five-subunit complex that is part of the processive eukaryotic DNA polymerase holoenzymes. Caspase-3 cleaves PARP at a DEVD-G motif present in the 140 kDa subunit of RF-C (RFC140) and evolutionarily conserved. We show that cleavage of RFC140 during Fas-mediated apoptosis in Jurkat cells and lymphocytes results in generation of multiple fragments. Cleavage is inhibited by the caspase-3-like protease inhibitor Ac-DEVD-CHO but not the caspase-1/ICE-type protease inhibitor Ac-YVAD-CHO. In addition, recombinant caspase-3 cleaves RFC140 in vitro at least at three different sites in the C-terminal half of the protein. Using amino-terminal microsequencing of radioactive fragments, we identified three sites: DEVD723G, DLVD922S and IETD1117A. We did not detect cleavage of small subunits of RF-C of 36, 37, 38 and 40 kDa by recombinant caspase-3 or by apoptotic Jurkat cell lysates. Cleavage of RFC140 during apoptosis inactivates its function in DNA replication and generates truncated forms that further inhibit DNA replication. These results identify RFC140 as a critical target for caspase-3-like proteases and suggest that caspases could mediate cell cycle arrest.     

A key role for replication factor C in DNA replication checkpoint function in fission yeast.

Replication factor C (RF-C) is a five subunit DNA polymerase (Pol) delta/straightepsilon accessory factor required at the replication fork for loading the essential processivity factor PCNA onto the 3'-ends of nascent DNA strands. Here we describe the genetic analysis of the rfc2 +gene of the fission yeast Schizosaccharomyces pombe encoding a structural homologue of the budding yeast Rfc2p and human hRFC37 proteins. Deletion of the rfc2 + gene from the chromosome is lethal but does not result in the checkpoint-dependent cell cycle arrest seen in cells deleted for the gene encoding PCNA or for those genes encoding subunits of either Pol delta or Pol straightepsilon. Instead, rfc2 Delta cells proceed into mitosis with incompletely replicated DNA, indicating that the DNA replication checkpoint is inactive under these conditions. Taken together with recent results, these observations suggest a simple model in which assembly of the RF-C complex onto the 3'-end of the nascent RNA-DNA primer is the last step required for the establishment of a checkpoint-competent state.     

Saccharomyces cerevisiae replication factor C. I. Purification and characterization of its ATPase activity.

Saccharomyces cerevisiae replication factor C (RF-C) was purified 25,000-fold from a protease-deficient strain of yeast. RF-C is a complex of 6 subunits of 130, 86, 41, 40, 37, and 27 kDa. None of the subunits are related through proteolysis or differential phosphorylation. The assay for RF-C used as a substrate single-stranded DNA binding protein-coated singly primed single-stranded mp 18 DNA. This DNA was poorly replicated by yeast DNA polymerase delta with or without its cofactor proliferating cell nuclear antigen (PCNA). In the presence of RF-C, however, replication of the template proceeded efficiently when both ATP and PCNA were present as well. Formation of this replication-proficient complex of DNA polymerase delta required an input of one to two molecules of PCNA per replicated DNA molecule. DNA polymerase epsilon also formed an ATP-dependent complex with PCNA and RF-C. RF-C has a DNA-dependent ATPase activity, equally active on single-stranded and primed single-stranded mp18 DNA. Addition of PCNA stimulated the ATPase of RF-C on primed but not on unprimed DNA, indicating that the increase in ATPase was due to PCNA-enhanced binding of RF-C to the primer terminus. Calf thymus PCNA also stimulated the ATPase activity of yeast RF-C and participated in holoenzyme formation with DNA polymerase delta. These results attest to the structural and functional homology between yeast and mammalian cells for these components of the replication machinery.     

A unique organization of the protein subunits of the DNA polymerase clamp loader in the archaeon Methanobacterium thermoautotrophicum deltaH

Replication factor C (RFC, also called activator 1), in conjunction with proliferating cell nuclear antigen (PCNA), is responsible for processive DNA synthesis catalyzed by the eukaryotic replicative DNA polymerases delta and epsilon. Here we report the isolation and characterization of homologues of RFC and PCNA from the archaeon, Methanobacterium thermoautotrophicum DeltaH. In contrast to the five subunit RFC complex isolated from eukaryotic cells, the mthRFC contains only two subunits. The two genes encoding the RFC subunits called, mthRFC1 and mthRFC3, were cloned, and the proteins (54.4 and 36.8 kDa, respectively) were overexpressed in Escherichia coli and purified individually and as a complex. The gene encoding PCNA was also cloned, and the protein was purified after overexpression in E. coli. Based on sizing column elution and subunit composition, the mthRFC complex appears to be a hexamer consisting of two mthRFC1 protomers and four mthRFC3 protomers. Although mthRFC differs in organization from its eukaryotic counterpart, it was shown to be functionally similar to eukaryotic RFC in: (i) catalyzing DNA-dependent ATP hydrolysis; (ii) binding preferentially to DNA primer ends; (iii) loading mthPCNA onto singly nicked circular DNA; and (iv) supporting mthPolB-catalyzed PCNA-dependent DNA chain elongation. The importance and roles of RFC and PCNA in M. thermoautotrophicum DeltaH replication are discussed.     

The RFC2 Gene, Encoding the Third-Largest Subunit of the Replication Factor C Complex, Is Required for an S-Phase Checkpoint in Saccharomyces cerevisiae

Replication factor C (RF-C), an auxiliary factor for DNA polymerases δ and , is a multiprotein complex consisting of five different polypeptides. It recognizes a primer on a template DNA, binds to a primer terminus, and helps load proliferating cell nuclear antigen onto the DNA template. The RFC2 gene encodes the third-largest subunit of the RF-C complex. To elucidate the role of this subunit in DNA metabolism, we isolated a thermosensitive mutation (rfc2-1) in the RFC2 gene. It was shown that mutant cells having the rfc2-1 mutation exhibit (i) temperature-sensitive cell growth; (ii) defects in the integrity of chromosomal DNA at restrictive temperatures; (iii) progression through cell cycle without definitive terminal morphology and rapid loss of cell viability at restrictive temperatures; (iv) sensitivity to hydroxyurea, methyl methanesulfonate, and UV light; and (v) increased rate of spontaneous mitotic recombination and chromosome loss. These phenotypes of the mutant suggest that the RFC2 gene product is required not only for chromosomal DNA replication but also for a cell cycle checkpoint. It was also shown that the rfc2-1 mutation is synthetically lethal with either the cdc44-1 or rfc5-1 mutation and that the restrictive temperature of rfc2-1 mutant cells can be lowered by combining either with the cdc2-2 or pol2-11 mutation. Finally, it was shown that the temperature-sensitive cell growth phenotype and checkpoint defect of the rfc2-1 mutation can be suppressed by a multicopy plasmid containing the RFC5 gene. These results suggest that the RFC2 gene product interacts with the CDC44/RFC1 and RFC5 gene products in the RF-C complex and with both DNA polymerases δ and during chromosomal DNA replication.     

RFCCtf18 and the Swi1-Swi3 complex function in separate and redundant pathways required for the stabilization of replication forks to facilitate sister chromatid cohesion in Schizosaccharomyces pombe

Sister chromatid cohesion is established during S phase near the replication fork. However, how DNA replication is coordinated with chromosomal cohesion pathway is largely unknown. Here, we report studies of fission yeast Ctf18, a subunit of the RFC(Ctf18) replication factor C complex, and Chl1, a putative DNA helicase. We show that RFC(Ctf18) is essential in the absence of the Swi1-Swi3 replication fork protection complex required for the S phase stress response. Loss of Ctf18 leads to an increased sensitivity to S phase stressing agents, a decreased level of Cds1 kinase activity, and accumulation of DNA damage during S phase. Ctf18 associates with chromatin during S phase, and it is required for the proper resumption of replication after fork arrest. We also show that chl1Delta is synthetically lethal with ctf18Delta and that a dosage increase of chl1(+) rescues sensitivities of swi1Delta to S phase stressing agents, indicating that Chl1 is involved in the S phase stress response. Finally, we demonstrate that inactivation of Ctf18, Chl1, or Swi1-Swi3 leads to defective centromere cohesion, suggesting the role of these proteins in chromosome segregation. We propose that RFC(Ctf18) and the Swi1-Swi3 complex function in separate and redundant pathways essential for replication fork stabilization to facilitate sister chromatid cohesion in fission yeast.     

Cloning and characterization of hCTF18, hCTF8, and hDCC1. Human homologs of a Saccharomyces cerevisiae complex involved in sister chromatid cohesion establishment

A growing body of evidence suggests that establishment of sister chromatid cohesion is dependent on replication fork passage over a precohesion area. In Saccharomyces cerevisiae, this process involves an alternative replication factor C (RFC) complex that contains the four small RFC subunits as well as CTF18, CTF8, and DCC1. Here, we show that an evolutionarily conserved homologous complex exists in the nucleus of human cells. We demonstrate that hCTF18, hCTF8, and hDCC1 interact with each other as well as with the p38 subunit of RFC. This alternative RFC-containing complex interacts with proliferating cell nuclear antigen but not with the Rad9/Rad1/Hus1 complex, a proliferating cell nuclear antigen-like clamp involved in the DNA damage response. hCTF18 preferentially binds chromatin during S phase, suggesting a role during replication. Our data provide evidence for the existence of an alternative RFC complex with a probable role in mammalian sister chromatid cohesion establishment.     

The Elg1 replication factor C-like complex: a novel guardian of genome stability

The remarkable stability of the eukaryotic genome is achieved by the activity of many overlapping surveillance and repair mechanism. Two protein complexes with resemblance to replication factor C (RFC) have been recently described, that play important roles in maintaining the stability of the genome. These RFC-like complexes (RLCs) share four common subunits (Rfc2-5) and each carry a unique large subunit (Rad24 or Ctf18) replacing the Rfc1 subunit of the replication complex. Work in several laboratories has recently uncovered a novel yeast gene, ELG1, which seems to play a central role in keeping the genome stable. elg1 mutants exhibit increased rates of spontaneous recombination and gross chromosomal rearrangements during vegetative growth. In addition, they lose chromosomes at an enhanced rate, show hyper-transposition of natural repeated elements and exhibit elongated telomeres. The Elg1 protein also associates with the Rfc2-5 subunits of replication factor C (RFC) to form a third RFC-like complex (RLC). Genetic and biochemical data indicate that the Elg1, Ctf18 and Rad24 RLCs work in three separate pathways important for maintaining the integrity of the genome and for coping with various genomic stresses. ELG1 is evolutionarily conserved and may play an important role in preventing the onset of cancer in humans. The Elg1 function is thus clearly required for maintaining genome stability during normal growth, and its absence has severe genetic consequences.     

Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion

CTF4 and CTF18 are required for high-fidelity chromosome segregation. Both exhibit genetic and physical ties to replication fork constituents. We find that absence of either CTF4 or CTF18 causes sister chromatid cohesion failure and leads to a preanaphase accumulation of cells that depends on the spindle assembly checkpoint. The physical and genetic interactions between CTF4, CTF18, and core components of replication fork complexes observed in this study and others suggest that both gene products act in association with the replication fork to facilitate sister chromatid cohesion. We find that Ctf18p, an RFC1-like protein, directly interacts with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. However, Ctf18p is not a component of biochemically purified proliferating cell nuclear antigen loading RF-C, suggesting the presence of a discrete complex containing Ctf18p, Rfc2p, Rfc3p, Rfc4p, and Rfc5p. Recent identification and characterization of the budding yeast polymerase kappa, encoded by TRF4, strongly supports a hypothesis that the DNA replication machinery is required for proper sister chromatid cohesion. Analogous to the polymerase switching role of the bacterial and human RF-C complexes, we propose that budding yeast RF-C(CTF18) may be involved in a polymerase switch event that facilities sister chromatid cohesion. The requirement for CTF4 and CTF18 in robust cohesion identifies novel roles for replication accessory proteins in this process.     

Characterization of the five replication factor C genes of Saccharomyces cerevisiae.

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.     

Fission Yeast Cdc24 Is a Replication Factor C- and Proliferating Cell Nuclear Antigen-Interacting Factor Essential for S-Phase Completion

At the nonpermissive temperature the fission yeast cdc24-M38 mutant arrests in the cell cycle with incomplete DNA replication as indicated by pulsed-field gel electrophoresis. The cdc24⁺ gene encodes a 501-amino-acid protein with no significant homology to any known proteins. The temperature-sensitive cdc24 mutant is effectively rescued by pcn1⁺, rfc1⁺ (a fission yeast homologue of RFC1), and hhp1⁺, which encode the proliferating cell nuclear antigen (PCNA), the large subunit of replication factor C (RFC), and a casein kinase I involved in DNA damage repair, respectively. The Cdc24 protein binds PCNA and RFC1 in vivo, and the domains essential for Cdc24 function and for RFC1 and PCNA binding colocalize in the N-terminal two-thirds of the molecule. In addition, cdc24⁺ genetically interacts with the gene encoding the catalytic subunit of DNA polymerase , which is stimulated by PCNA and RFC, and with those encoding the fission yeast counterparts of Mcm2, Mcm4, and Mcm10. These results indicate that Cdc24 is an RFC- and PCNA-interacting factor required for DNA replication and might serve as a target for regulation.     

Chl12 (Ctf18) forms a novel replication factor C-related complex and functions redundantly with Rad24 in the DNA replication checkpoint pathway

RAD24 has been identified as a gene essential for the DNA damage checkpoint in budding yeast. Rad24 is structurally related to subunits of the replication factor C (RFC) complex, and forms an RFC-related complex with Rfc2, Rfc3, Rfc4, and Rfc5. The rad24Delta mutation enhances the defect of rfc5-1 in the DNA replication block checkpoint, implicating RAD24 in this checkpoint. CHL12 (also called CTF18) encodes a protein that is structurally related to the Rad24 and RFC proteins. We show here that although neither chl12Delta nor rad24Delta single mutants are defective, chl12Delta rad24Delta double mutants become defective in the replication block checkpoint. We also show that Chl12 interacts physically with Rfc2, Rfc3, Rfc4, and Rfc5 and forms an RFC-related complex which is distinct from the RFC and RAD24 complexes. Our results suggest that Chl12 forms a novel RFC-related complex and functions redundantly with Rad24 in the DNA replication block checkpoint.     

A conserved domain of the large subunit of replication factor C binds PCNA and acts like a dominant negative inhibitor of DNA replication in mammalian cells.

Replication factor C (RF-C), a complex of five polypeptides, is essential for cell-free SV40 origin-dependent DNA replication and viability in yeast. The cDNA encoding the large subunit of human RF-C (RF-Cp145) was cloned in a Southwestern screen. Using deletion mutants of RF-Cp145 we have mapped the DNA binding domain of RF-Cp145 to amino acid residues 369-480. This domain is conserved among both prokaryotic DNA ligases and eukaryotic poly(ADP-ribose) polymerases and is absent in other subunits of RF-C. The PCNA binding domain maps to amino acid residues 481-728 and is conserved in all five subunits of RF-C. The PCNA binding domain of RF-Cp145 inhibits several functions of RF-C, such as: (i) in vitro DNA replication of SV40 origin-containing DNA; (ii) RF-C-dependent loading of PCNA onto DNA; and (iii) RF-C-dependent DNA elongation. The PCNA binding domain of RF-Cp145 localizes to the nucleus and inhibits DNA synthesis in transfected mammalian cells. In contrast, the DNA binding domain of RF-Cp145 does not inhibit DNA synthesis in vitro or in vivo. We therefore conclude that amino acid residues 481-728 of human RF-Cp145 are critical and act as a dominant negative mutant of RF-C function in DNA replication in vivo.     

Allele-specific interactions between the yeast RFC1 and RFC5 genes suggest a basis for RFC subunit-subunit interactions

Replication factor C (RFC) is an essential, multi-subunit ATPase that functions in DNA replication, DNA repair, and DNA metabolism-related checkpoints. In order to investigate how the individual RFC subunits contribute to these functions in vivo, we undertook a genetic analysis of RFC genes from budding yeast. We isolated and characterized mutations in the RFC5 gene that could suppress the cold-sensitive phenotype of rfc1-1 mutants. Analysis of the RFC5 suppressors revealed that they could not suppress the elongated telomere phenotype, the sensitivity to DNA damaging agents, or the mutator phenotype of rfc1-1 mutants. Unlike the checkpoint-defective rfc5-1 mutation, the RFC5 suppressor mutations did not interfere with the methylmethane sulfonate- or hydroxyurea-induced phosphorylation of Rad53p. The Rfc5p suppressor substitutions mapped to amino acid positions in the conserved RFC box motifs IV-VII. Comparisons of the structures of related RFC box-containing proteins suggest that these RFC motifs may function to coordinate interactions between neighboring subunits of multi-subunit ATPases.     

Rfc4 interacts with Rpa1 and is required for both DNA replication and DNA damage checkpoints in Saccharomyces cerevisiae

The large subunit of replication protein A (Rpa1) consists of three single-stranded DNA binding domains and an N-terminal domain (Rpa1N) of unknown function. To determine the essential role of this domain we searched for mutations that require wild-type Rpa1N for viability in yeast. A mutation in RFC4, encoding a small subunit of replication factor C (RFC), was found to display allele-specific interactions with mutations in the gene encoding Rpa1 (RFA1). Mutations that map to Rpa1N and confer sensitivity to the DNA synthesis inhibitor hydroxyurea, such as rfa1-t11, are lethal in combination with rfc4-2. The rfc4-2 mutant itself is sensitive to hydroxyurea, and like rfc2 and rfc5 strains, it exhibits defects in the DNA replication block and intra-S checkpoints. RFC4 and the DNA damage checkpoint gene RAD24 were found to be epistatic with respect to DNA damage sensitivity. We show that the rfc4-2 mutant is defective in the G(1)/S DNA damage checkpoint response and that both the rfc4-2 and rfa1-t11 strains are defective in the G(2)/M DNA damage checkpoint. Thus, in addition to its essential role as part of the clamp loader in DNA replication, Rfc4 plays a role as a sensor in multiple DNA checkpoint pathways. Our results suggest that a physical interaction between Rfc4 and Rpa1N is required for both roles.     

A novel Rad24 checkpoint protein complex closely related to replication factor C

Rad24 functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae. Here, analysis of Rad24 in whole cell extracts demonstrated that its mass was considerably greater than its predicted molecular weight, suggesting that Rad24 is a component of a protein complex. The Rad24 complex was purified to homogeneity. In addition to Rad24, the complex included polypeptides of 40 kDa and 35 kDa. The 40 kDa species was found by mass spectrometry to contain Rfc2 and Rfc3, subunits of replication factor C (RFC), a five subunit protein that is required for the loading of polymerases onto DNA during replication and repair [3]. We hypothesised that other RFC subunits, all of which share sequence homologles with Rad24, might also be components of the Rad24 complex. Reciprocal co-immunoprecipitation studies were performed using extracts prepared from strains containing epitope-tagged RFC proteins. These experiments showed that the small RFC proteins, Rfc2, Rfc3, Rfc4 and Rfc5, interacted with Rad24, whereas the Rfc1 subunit did not. We suggest that this RFC-like Rad24 complex may function as a structure-specific sensor in the DNA damage checkpoint pathway.     

The second subunit of DNA polymerase III (delta) is encoded by the HYS2 gene in Saccharomyces cerevisiae.

DNA polymerase III (delta) of Saccharomyces cerevisiae is purified as a complex of at least two polypeptides with molecular masses of 125 and 55 kDa as judged by SDS-PAGE. In this paper we determine partial amino acid sequences of the 125 and 55 kDa polypeptides and find that they match parts of the amino acid sequences predicted from the nucleotide sequence of the CDC2 and HYS2 genes respectively. We also show by Western blotting that Hys2 protein co-purifies with DNA polymerase III activity as well as Cdc2 polypeptide. The complex form of DNA polymerase III activity could not be detected in thermosensitive hys2 mutant cell extracts, although another form of DNA polymerase III was found. This form of DNA polymerase III, which could also be detected in wild-type extracts, was not associated with Hys2 protein and was not stimulated by addition of proliferating cell nuclear antigen (PCNA), replication factor A (RF-A) or replication factor C (RF-C). The temperature-sensitive growth phenotype of hys2-1 and hys2-2 mutations could be suppressed by the CDC2 gene on a multicopy plasmid. These data suggest that the 55 kDa polypeptide encoded by the HYS2 gene is one of the subunits of DNA polymerase III complex in S.cerevisiae and is required for highly processive DNA synthesis catalyzed by DNA polymerase III in the presence of PCNA, RF-A and RF-C.