ATP utilization by yeast replication factor C. III. The ATP-binding domains of Rfc2, Rfc3, and Rfc4 are essential for DNA recognition and clamp loading.

The conserved lysine in the Walker A motif of the ATP-binding domain encoded by the yeast RFC1, RFC2, RFC3, and RFC4 genes was mutated to glutamic acid. Complexes of replication factor C with a N-terminal truncation (Delta2-273) of the Rfc1 subunit (RFC) containing a single mutant subunit were overproduced in Escherichia coli for biochemical analysis. All of the mutant RFC complexes were capable of interacting with PCNA. Complexes containing a rfc1-K359E mutation were similar to wild type in replication activity and ATPase activity; however, the mutant complex showed increased susceptibility to proteolysis. In contrast, complexes containing either a rfc2-K71E mutation or a rfc3-K59E mutation were severely impaired in ATPase and clamp loading activity. In addition to their defects in ATP hydrolysis, these complexes were defective for DNA binding. A mutant complex containing the rfc4-K55E mutation performed as well as a wild type complex in clamp loading, but only at very high ATP concentrations. Mutant RFC complexes containing rfc2-K71R or rfc3-K59R, carrying a conservative lysine --> arginine mutation, had much milder clamp loading defects that could be partially (rfc2-K71R) or completely (rfc3-K59R) suppressed at high ATP concentrations.     

Rfc5, in cooperation with rad24, controls DNA damage checkpoints throughout the cell cycle in Saccharomyces cerevisiae

RAD24 and RFC5 are required for DNA damage checkpoint control in the budding yeast Saccharomyces cerevisiae. Rad24 is structurally related to replication factor C (RFC) subunits and associates with RFC subunits Rfc2, Rfc3, Rfc4, and Rfc5. rad24Delta mutants are defective in all the G(1)-, S-, and G(2)/M-phase DNA damage checkpoints, whereas the rfc5-1 mutant is impaired only in the S-phase DNA damage checkpoint. Both the RFC subunits and Rad24 contain a consensus sequence for nucleoside triphosphate (NTP) binding. To determine whether the NTP-binding motif is important for Rad24 function, we mutated the conserved lysine(115) residue in this motif. The rad24-K115E mutation, which changes lysine to glutamate, confers a complete loss-of-function phenotype, while the rad24-K115R mutation, which changes lysine to arginine, shows no apparent phenotype. Although neither rfc5-1 nor rad24-K115R single mutants are defective in the G(1)- and G(2)/M-phase DNA damage checkpoints, rfc5-1 rad24-K115R double mutants become defective in these checkpoints. Coimmunoprecipitation experiments revealed that Rad24(K115R) fails to interact with the RFC proteins in rfc5-1 mutants. Together, these results indicate that RFC5, like RAD24, functions in all the G(1)-, S- and G(2)/M-phase DNA damage checkpoints and suggest that the interaction of Rad24 with the RFC proteins is essential for DNA damage checkpoint control.     

Requirement for ATP by the DNA damage checkpoint clamp loader

The DNA damage clamp loader replication factor C (RFC-Rad24) consists of the Rad24 protein and the four small Rfc2-5 subunits of RFC. This complex loads the heterotrimeric DNA damage clamp consisting of Rad17, Mec3, and Ddc1 (Rad17/3/1) onto partial duplex DNA in an ATP-dependent manner. Interactions between the clamp loader and the clamp have been proposed to mirror those of the replication clamp loader RFC and the sliding clamp proliferating cell nuclear antigen (PCNA). In that system, three ATP molecules bound to the Rfc2, Rfc3, and Rfc4 subunits are necessary and sufficient for efficient loading of PCNA, whereas ATP binding to Rfc1 is not required. In contrast, in this study, we show that mutant RFC-Rad24 with a rad24-K115E mutation in the ATP-binding domain of Rad24 shows defects in the ATPase of the complex and is defective for interaction with Rad17/3/1 and for loading of the checkpoint clamp. A similar defect was measured with a mutant RFC-Rad24 clamp loader carrying a rfc4K55R ATP-binding mutation, whereas the rfc4K55E clamp loader showed partial loading activity, in agreement with genetic studies of these mutants. These studies show that ATP utilization by the checkpoint clamp/clamp loader system is effectively different from that by the structurally analogous replication system.     

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.     

Replication factor C3 of Schizosaccharomyces pombe, a small subunit of replication factor C complex, plays a role in both replication and damage checkpoints

We report here the isolation and functional analysis of the rfc3(+) gene of Schizosaccharomyces pombe, which encodes the third subunit of replication factor C (RFC3). Because the rfc3(+) gene was essential for growth, we isolated temperature-sensitive mutants. One of the mutants, rfc3-1, showed aberrant mitosis with fragmented or unevenly separated chromosomes at the restrictive temperature. In this mutant protein, arginine 216 was replaced by tryptophan. Pulsed-field gel electrophoresis suggested that rfc3-1 cells had defects in DNA replication. rfc3-1 cells were sensitive to hydroxyurea, methanesulfonate (MMS), and gamma and UV irradiation even at the permissive temperature, and the viabilities after these treatments were decreased. Using cells synchronized in early G2 by centrifugal elutriation, we found that the replication checkpoint triggered by hydroxyurea and the DNA damage checkpoint caused by MMS and gamma irradiation were impaired in rfc3-1 cells. Association of Rfc3 and Rad17 in vivo and a significant reduction of the phosphorylated form of Chk1 in rfc3-1 cells after treatments with MMS and gamma or UV irradiation suggested that the checkpoint signal emitted by Rfc3 is linked to the downstream checkpoint machinery via Rad17 and Chk1. From these results, we conclude that rfc3(+) is required not only for DNA replication but also for replication and damage checkpoint controls, probably functioning as a checkpoint sensor.     

Elg1 forms an alternative RFC complex important for DNA replication and genome integrity

Genome-wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork-processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2-5. This complex is distinct from the DNA replication RFC, the DNA damage checkpoint RFC and the sister chromatid cohesion RFC. As expected from its genetic interactions, elg1 mutants are sensitive to DNA damage. Elg1 is redundant with Rad24 in the DNA damage response and contributes to activation of the checkpoint kinase Rad53. We find that elg1 mutants display DNA replication defects and genome instability, including increased recombination and mutation frequencies, and minichromosome maintenance defects. Mutants in elg1 show genetic interactions with pathways required for processing of stalled replication forks, and are defective in recovery from DNA damage during S phase. We propose that Elg1-RFC functions both in normal DNA replication and in the DNA damage response.     

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.     

The product of Saccharomyces cerevisiae WHIP/MGS1, a gene related to replication factor C genes,interacts functionally with DNA polymerase delta

The Saccharomyces cerevisiae gene WHIP/ MGS1 encodes a protein related to the subunits of Replication Factor C (RFC). We found that the RFC-like motifs in Whip/Mgs1 are essential for its function. Furthermore, by screening for synthetic dosage lethality, we have shown that overexpression of MGS1 causes lethality in combination with mutations in genes that encode replication proteins such as DNA polymerase delta, RFC, PCNA and RPA. Moreover, loss of MGS1 function interferes with the ability of multicopy PCNA to suppress the replication defect of the rfc5-1 mutant. At permissive temperatures, deletion of MGS1 suppresses the hydroxyurea (HU) sensitivity of pol31 and pol32 mutants, which bear mutations in the smaller subunits of DNA polymerase delta, and at semipermissive and non-permissive temperatures mgs1delta partially alleviates the growth defects of the pol31 mutant. We also report that the growth defect and HU sensitivity of the pol31 mutant are suppressed by mms2delta and rad18delta mutations. We suggest that Mgs1 interacts with the DNA replication machinery to modulate the function of DNA polymerase delta during replication or replication-associated repair, and influences the choice of the pathway employed for replication fork reactivation. Possible roles of Mgs1, DNA polymerase delta, Rad18 and Mms2 in replication and replication fork restart 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.     

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.     

Dominant mutations in three different subunits of replication factor C suppress replication defects in yeast PCNA mutants

To identify proteins that interact with the yeast proliferating cell nuclear antigen (PCNA), we used a genetic approach to isolate mutations that compensate for the defects in cold-sensitive (Cs(-)) mutants of yeast PCNA (POL30). Because the cocrystal structure of human PCNA and a p21(WAF1/CIP1) peptide shows that the interdomain region of PCNA is a site of p21 interaction, we specifically looked for new mutations that suppress mutations in the equivalent region of yeast PCNA. In independent screens using three different Cs(-) mutants, we identified spontaneously arising dominant suppressor mutations in the RFC3 gene. In addition, dominant suppressor mutations were identified in the RFC1 and RFC2 genes using a single pol30 mutant. An intimate association between PCNA and RFC1p, RFC2p, and RFC3p is suggested by the allele-restricted suppression of 10 different pol30 alleles by the RFC suppressors. RFC1, RFC2, and RFC3 encode three of the five subunits of the replication factor C complex, which is required to load PCNA onto DNA in reconstituted DNA replication reactions. Genomic sequencing reveals a common region in RFC1p, RFC2p, and RFC3p that is important for the functional interaction with PCNA. Biochemical analysis of the wild type and mutant PCNA and RFC3 proteins shows that mutant RFC3p enhances the production of long DNA products in pol delta-dependent DNA synthesis, which is consistent with an increase in processivity.     

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.     

Methylations of histone H3 lysine 9 and lysine 36 are functionally linked to DNA replication checkpoint control in fission yeast

Recently, histone H4 lysine 20 and H3 lysine 79 methylations were functionally linked to DNA damage checkpoint. The crosstalk between histone methylation and the S-M checkpoint, however, has remained unclear. Here, we show that H3 lysine 9 (K9) and lysine 36 (K36) methylations catalyzed by two histone methyltransferases Clr4 and Set2 are involved in hydroxyurea (HU)-induced replication checkpoint. The clr4-set2 double mutants besides histone H3-K9 and K36 double mutants exhibited HU-sensitivity, a defective HU-induced S-M checkpoint, and a significant reduction of HU-induced phosphorylation of Cdc2. Intriguingly, the clr4-set2 double mutations impaired the HU-induced accumulation of a mitotic inhibitor Mik1. Double mutants in Alp13 and Swi6, which can specifically bind to H3-K36 and K9 methylations, exhibited phenotypes similar to those of the clr4-set2 mutants. Together, these findings suggest that methylations of histone H3-K9 and K36 by Clr4 and Set2 are functionally linked to DNA replication checkpoint via accumulation of Mik1.     

Functional and Physical Interaction between Rad24 and Rfc5 in the Yeast Checkpoint Pathways

The RFC5 gene encodes a small subunit of replication factor C (RFC) complex in Saccharomyces cerevisiae and has been shown to be required for the checkpoints which respond to replication block and DNA damage. Here we describe the isolation of RAD24, known to play a role in the DNA damage checkpoint, as a dosage-dependent suppressor of rfc5-1. RAD24 overexpression suppresses the sensitivity of rfc5-1 cells to DNA-damaging agents and the defect in DNA damage-induced Rad53 phosphorylation. Rad24, like Rfc5, is required for the regulation of Rad53 phosphorylation in response to DNA damage. The Rad24 protein, which is structurally related to the RFC subunits, interacts physically with RFC subunits Rfc2 and Rfc5 and cosediments with Rfc5. Although the rad24Δ mutation alone does not cause a defect in the replication block checkpoint, it does enhance the defect in rfc5-1 mutants. Furthermore, overexpression of RAD24 suppresses the rfc5-1 defect in the replication block checkpoint. Taken together, our results demonstrate a physical and functional interaction between Rad24 and Rfc5 in the checkpoint pathways.     

拟南芥复制因子C亚基1在胚胎发生中起重要作用

复制因子C包含1个大亚基和4个小亚基,在DNA复制、损伤修复和细胞增殖中起重要作用,拟南芥复制因子C亚基1(AtRFC1)是人类复制因子C大亚基p140的同源蛋白。在对3个复制因子C亚基1的T-DNA插入突变株系rfc1-1、rfc1-2和rfc1-3的检验中,证实插入位点分别位于第16、19号外显子和启动子区域。T-DNA在外显子中的插入突变引起胚胎发育异常并导致胚胎和种子败育。将野生型拟南芥复制因子C亚基1基因转化到突变株系rfc1-1和rfc1-2后恢复了突变株的野生型表型,证明胚胎发生异常表型是由拟南芥复制因子C亚基1基因突变所引起的,AtRFC1在拟南芥胚胎发生中起重要作用。     

Arabidopsis replication factor C4 is critical for DNA replication during the mitotic cell cycle

Replication factor C (RFC) is a conserved eukaryotic complex consisting of RFC1/2/3/4/5. It plays important roles in DNA replication and the cell cycle in yeast and fruit fly. However, it is not very clear how RFC subunits function in higher plants, except for the Arabidopsis (At) subunits AtRFC1 and AtRFC3. In this study, we investigated the functions of AtRFC4 and found that loss of function of AtRFC4 led to an early sporophyte lethality that initiated as early as the elongated zygote stage, all defective embryos arrested at the two‐ to four‐cell embryo proper stage, and the endosperm possessed six to eight free nuclei. Complementation of rfc4‐1/+ with AtRFC4 expression driven through the embryo‐specific DD45pro and ABI3pro or the endosperm‐specific FIS2pro could not completely restore the defective embryo or endosperm, whereas a combination of these three promoters in rfc4‐1/+ enabled the aborted ovules to develop into viable seeds. This suggests that AtRFC4 functions simultaneously in endosperm and embryo and that the proliferation of endosperm is critical for embryo maturation. Assays of DNA content in rfc4‐1/+ verified that DNA replication was disrupted in endosperm and embryo, resulting in blocked mitosis. Moreover, we observed a decreased proportion of late S‐phase and M‐phase cells in the rfc4‐1/–^{FIS2;DD45;ABI3pro::AtRFC4} seedlings, suggesting that incomplete DNA replication triggered cell cycle arrest in cells of the root apical meristem. Therefore, we conclude that AtRFC4 is a crucial gene for DNA replication.     

Contrasting effects of Elg1-RFC and Ctf18-RFC inactivation in the absence of fully functional RFC in fission yeast

Proliferating cell nuclear antigen loading onto DNA by replication factor C (RFC) is a key step in eukaryotic DNA replication and repair processes. In this study, the C-terminal domain (CTD) of the large subunit of fission yeast RFC is shown to be essential for its function in vivo. Cells carrying a temperature-sensitive mutation in the CTD, rfc1-44, arrest with incompletely replicated chromosomes, are sensitive to DNA damaging agents, are synthetically lethal with other DNA replication mutants, and can be suppressed by mutations in rfc5. To assess the contribution of the RFC-like complexes Elg1–RFC and Ctf18–RFC to the viability of rfc1-44, genes encoding the large subunits of these complexes have been deleted and overexpressed. Inactivation of Ctf18–RFC by the deletion of ctf18⁺, dcc1⁺ or ctf8⁺ is lethal in an rfc1-44 background showing that full Ctf18–RFC function is required in the absence of fully functional RFC. In contrast, rfc1-44 elg1Δ cells are viable and overproduction of Elg1 in rfc1-44 is lethal, suggesting that Elg1–RFC plays a negative role when RFC function is inhibited. Consistent with this, the deletion of elg1⁺ is shown to restore viability to rfc1-44 ctf18Δ cells.     

Srs2 overexpression reveals a helicase-independent role at replication forks that requires diverse cell functions

Srs2 is a 3'-5' DNA helicase that regulates many aspects of DNA metabolism in Saccharomyces cerevisiae. It is best known for its ability to counteract homologous recombination by dismantling Rad51 filaments, but is also involved in checkpoint activation, adaptation and recovery, and in resolution of late recombination intermediates. To further address its biological roles and uncover new genetic interactions, we examined the consequences of overexpressing SRS2 as well as two helicase-dead mutants, srs2-K41A and srs2-K41R, in the collection of 4827 yeast haploid deletion mutants. We identified 274 genes affecting a large variety of cellular functions that are required for cell growth when SRS2 or its mutants are overexpressed. Further analysis of these interactions reveals that Srs2 acts independently of its helicase function at replication forks likely through its recruitment by the sumoylated PCNA replication clamp. This helicase-independent function is responsible for the negative interactions with DNA metabolism genes and for the toxicity of SRS2 overexpression in many of the diverse cellular pathways revealed in our screens.     

A requirement for recombinational repair in Saccharomyces cerevisiae is caused by DNA replication defects of mec1 mutants.

To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.