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.     

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.     

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.     

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.     

ATP utilization by yeast replication factor C. IV. RFC ATP-binding mutants show defects in DNA replication, DNA repair, and checkpoint regulation.

Replication factor C is required to load proliferating cell nuclear antigen onto primer-template junctions, using the energy of ATP hydrolysis. Four of the five RFC genes have consensus ATP-binding motifs. To determine the relative importance of these sites for proper DNA metabolism in the cell, the conserved lysine in the Walker A motif of RFC1, RFC2, RFC3, or RFC4 was mutated to either arginine or glutamic acid. Arginine mutations in all RFC genes tested permitted cell growth, although poor growth was observed for rfc2-K71R. A glutamic acid substitution resulted in lethality in RFC2 and RFC3 but not in RFC1 or RFC4. Most double mutants combining mutations in two RFC genes were inviable. Except for the rfc1-K359R and rfc4-K55E mutants, which were phenotypically similar to wild type in every assay, the mutants were sensitive to DNA-damaging agents. The rfc2-K71R and rfc4-K55R mutants show checkpoint defects, most likely in the intra-S phase checkpoint. Regulation of the damage-inducible RNR3 promoter was impaired in these mutants, and phosphorylation of Rad53p in response to DNA damage was specifically defective when cells were in S phase. No dramatic defects in telomere length regulation were detected in the mutants. These data demonstrate that the ATP binding function of RFC2 is important for both DNA replication and checkpoint function and, for the first time, that RFC4 also plays a role in checkpoint regulation.     

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.     

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.     

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.     

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.     

Mechanical link between cohesion establishment and DNA replication: Ctf7p/Eco1p,a cohesion establishment factor,associates with three different replication factor C complexes

CTF7/ECO1 is an essential yeast gene required for the establishment of sister chromatid cohesion. The findings that CTF7/ECO1, POL30 (PCNA), and CHL12/CTF18 (a replication factor C [RFC] homolog) genetically interact provided the first evidence that the processes of cohesion establishment and DNA replication are intimately coupled-a link now confirmed by other studies. To date, however, it is unknown how Ctf7p/Eco1p function is coupled to DNA replication or whether Ctf7p/Eco1p physically associates with any components of the DNA replication machinery. Here, we report that Ctf7p/Eco1p associates with proteins that perform partially redundant functions in DNA replication. Chl12p/Ctf18p combines with Rfc2p to Rfc5p to form one of three independent RFC complexes. By chromatographic methods, Ctf7p/Eco1p was found to associate with Chl12/Ctf18p and with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. The association between Ctf7p/Eco1p and this RFC complex is biologically relevant in that (i) Ctf7p/Eco1p cosediments with Chl12p/Ctf18p in vivo and (ii) rfc5-1 mutant cells exhibit precocious sister separation. Previous studies revealed that Rfc1p or Rad24p associates with Rfc2p to Rfc5p to form two other RFC complexes independent of Ctf18p-RFC complexes. These Rfc1p-RFC and Rad24p-RFC complexes function in DNA replication or repair and DNA damage checkpoint pathways. Importantly, Ctf7p/Eco1p also associates with Rfc1p and Rad24p, suggesting that these RFC complexes also play critical roles in cohesion establishment. The associations between Ctf7p/Eco1p and RFC subunits provide novel evidence regarding the physical linkage between cohesion establishment and DNA replication. Furthermore, the association of Ctf7p/Eco1p with each of three RFC complexes supplies new insights into the functional redundancy of RFC complexes in cohesion establishment.     

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.     

Fission yeast Rad17 associates with chromatin in response to aberrant genomic structures

Fission yeast checkpoint protein Rad17 is required for the DNA integrity checkpoint responses. A fraction of Rad17 is chromatin bound independent of the other checkpoint proteins throughout the cell cycle. Here we show that in response to DNA damage induced by either methyl methanesulfonate treatment or ionizing radiation, increased levels of Rad17 bind to chromatin. Following S-phase stall induced by hydroxyurea or a cdc22 mutation, the chromatin-bound Rad17 progressively dissociates from the chromatin. After S-phase arrest by hydroxyurea in cds1Delta or rad3Delta cells or by replication mutants, Rad17 remains chromatin bound. Rad17 is able to complex in vivo with an Rfc small subunit, Rfc2, but not with Rfc1. Furthermore, cells with rfc1Delta are checkpoint proficient, suggesting that Rfc1 does not have a role in checkpoint function. A checkpoint-defective mutant protein, Rad17(K118E), which has similar nuclear localization to that of the wild type, is unable to bind ATP and has reduced ability in chromatin binding. Mutant Rad17(K118E) protein also has reduced ability to complex with Rfc2, suggesting that Lys(118) of Rad17 plays a role in Rad17-Rfc small-subunit complex formation and chromatin association. However, in the rad17.K118E mutant cells, Cds1 can be activated by hydroxyurea. Together, these results suggest that Rad17 binds to chromatin in response to an aberrant genomic structure generated from DNA damage, replication mutant arrest, or hydroxyurea arrest in the absence of Cds1. Rad17 is not required to bind chromatin when genomic structures are protected by hydroxyurea-activated Cds1. The possible checkpoint events induced by chromatin-bound Rad17 are discussed.     

The human checkpoint protein hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9

DNA damage activates cell cycle checkpoints that prevent progression through the cell cycle. In yeast, the DNA damage checkpoint response is regulated by a series of genes that have mammalian homologs, including rad1, rad9, hus1, and rad17. On the basis of sequence homology, yeast and human Rad1, Rad9, and Hus1 protein homologs are predicted to structurally resemble the sliding clamp PCNA. Likewise, Rad17 homologs have extensive homology with replication factor C (RFC) subunits (p36, p37, p38, p40, and p140), which form a clamp loader for PCNA. These observations predict that Rad1, Hus1, and Rad9 might interact with Rad17 as a clamp-clamp loader pair during the DNA damage response. In this report, we demonstrate that endogenous human Rad17 (hRad17) interacts with the PCNA-related checkpoint proteins hRad1, hRad9, and hHus1. Mutational analysis of hRad1 and hRad17 demonstrates that this interaction has properties similar to the interaction between RFC and PCNA, a well characterized clamp-clamp loader pair. Moreover, we show that DNA damage affects the association of hRad17 with the clamp-like checkpoint proteins. Collectively, these data provide the first experimental evidence that hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9 in manner similar to the interaction between RFC and PCNA.     

Mechanism of proliferating cell nuclear antigen clamp opening by replication factor C

The eukaryotic replication factor C (RFC) clamp loader is an AAA+ spiral-shaped heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) clamp processivity factor on DNA. In this study, we examined the roles of individual RFC subunits in opening the PCNA clamp. Interestingly, Rfc1, which occupies the position analogous to the delta clamp-opening subunit in the Escherichia coli clamp loader, is not required to open PCNA. The Rfc5 subunit is required to open PCNA. Consistent with this result, Rfc2.3.4.5 and Rfc2.5 subassemblies are capable of opening and unloading PCNA from circular DNA. Rfc5 is positioned opposite the PCNA interface from Rfc1, and therefore, its action with Rfc2 in opening PCNA indicates that PCNA is opened from the opposite side of the interface that the E. coli delta wrench acts upon. This marks a significant departure in the mechanism of eukaryotic and prokaryotic clamp loaders. Interestingly, the Rad.RFC DNA damage checkpoint clamp loader unloads PCNA clamps from DNA. We propose that Rad.RFC may clear PCNA from DNA to facilitate shutdown of replication in the face of DNA damage.     

LCD1: an essential gene involved in checkpoint control and regulation of the MEC1 signalling pathway in Saccharomyces cerevisiae

We identified YDR499W as a Saccharomyces cerevisiae open reading frame with homology to several checkpoint proteins, including S. cerevisiae Rfc5p and Schizosaccharomyces pombe Rad26. Disruption of YDR499W (termed LCD1) results in lethality that is rescued by increasing cellular deoxyribonucleotide levels. Cells lacking LCD1 are very sensitive to a range of DNA-damaging agents, including UV irradiation, and to the inhibition of DNA replication. LCD1 is necessary for the phosphorylation and activation of Rad53p in response to DNA damage or DNA replication blocks, and for Chk1p activation in response to DNA damage. LCD1 is also required for efficient DNA damage-induced phosphorylation of Rad9p and for the association of Rad9p with the FHA2 domain of Rad53p after DNA damage. In addition, cells lacking LCD1 are completely defective in the G(1)/S and G(2)/M DNA damage checkpoints. Finally, we reveal that endogenous Mec1p co-immunoprecipitates with Lcd1p both before and after treatment with DNA-damaging agents. These results indicate that Lcd1p is a pivotal checkpoint regulator, involved in both the essential and checkpoint functions of the Mec1p pathway.     

The Saccharomyces cerevisiae checkpoint genes RAD9, CHK1 and PDS1 are required for elevated homologous recombination in a mec1 (ATR) hypomorphic mutant

Specific ataxia telangiectasia and Rad3-related (ATR) mutations confer higher frequencies of homologous recombination. The genetic requirements for hyper-recombination in ATR mutants are unknown. MEC1, the essential yeast ATR/ATM homolog, controls S and G2 checkpoints and the DNA damage-inducibility of genes after radiation exposure. Since the mec1-D (null) mutant is defective in both S and G2 checkpoints, we measured spontaneous and DNA damage-associated sister chromatid exchange (SCE), homolog (heteroallelic) recombination, and homology-directed translocations in the mec1-21 hypomorphic mutant, which is defective in the S phase checkpoint but retains some G2 checkpoint function. We observed a sixfold, tenfold and 30-fold higher rate of spontaneous SCE, heteroallelic recombination, and translocations, respectively, in mec1-21 mutants compared to wild type. The mec1-21 hyper-recombination was partially reduced in rad9, pds1, and chk1 mutants, and abolished in rad52 mutants, suggesting the hyper-recombination results from RAD52-dependent recombination pathway(s) that require G2 checkpoint functions. The HU and UV sensitivities of mec1-21 rad9 and mec1-21 rad52 were synergistically increased, compared to the single mutants, indicating that mec1-21, rad52 and rad9 mutants are defective in independent pathways for HU and UV resistance. G2-arrested mec1-21 rad9 cells exhibit more UV resistance than non-synchronized cells, indicating that one function of RAD9 in conferring UV resistance in mec1-21 is by triggering G2 arrest. We suggest that checkpoint genes that function in the RAD9-mediated pathway are required for either homologous recombination or DNA damage resistance in the S phase checkpoint mutant mec1-21.     

Fission yeast rad17: a homologue of budding yeast RAD24 that shares regions of sequence similarity with DNA polymerase accessory proteins.

Following DNA damage or a block to DNA synthesis, checkpoint pathways act to arrest mitosis and prevent the attempted segregation of damaged or unreplicated DNA. The rad17 locus of Schizosaccharomyces pombe is one of seven known radiation-sensitive (rad) loci which are absolutely required to prevent mitosis following DNA damage in fission yeast. Six of these (rad1, rad3, rad9, rad17, rad26 and hus1) are also required for the checkpoint which prevents mitosis from occurring before DNA replication is complete. We report here that the predicted rad17 gene product is a basic hydrophilic protein of 606 amino acids which contains five domains with sequence homology to replication factor C (RF-C)/activator 1 subunits. Western analysis and fusion with Green Fluorescent Protein indicate that the abundance and electrophoretic mobility of Rad17 is not significantly modified following a block to DNA synthesis or following DNA damage, and that Rad17 is localized in the nucleus. Rad17 function is not essential for growth, but is required for the function of the DNA structure-dependent checkpoints. Site-directed mutagenesis has been used to demonstrate the biological significance of the RF-C/activator 1-related domains. These studies have also defined an element of the radiation sensitivity caused by loss of Rad17 function which is not associated with the radiation-induced G2 arrest defect seen in the rad17.d null mutant cells.     

Yeast Rad52 and Rad51 recombination proteins define a second pathway of DNA damage assessment in response to a single double-strand break

Saccharomyces cells with a single unrepaired double-strand break adapt after checkpoint-mediated G(2)/M arrest. We have found that both Rad51 and Rad52 recombination proteins play key roles in adaptation. Cells lacking Rad51p fail to adapt, but deleting RAD52 suppresses rad51Delta. rad52Delta also suppresses adaptation defects of srs2Delta mutants but not those of yku70Delta or tid1Delta mutants. Neither rad54Delta nor rad55Delta affects adaptation. A Rad51 mutant that fails to interact with Rad52p is adaptation defective; conversely, a C-terminal truncation mutant of Rad52p, impaired in interaction with Rad51p, is also adaptation defective. In contrast, rad51-K191A, a mutation that abolishes recombination and results in a protein that does not bind to single-stranded DNA (ssDNA), supports adaptation, as do Rad51 mutants impaired in interaction with Rad54p or Rad55p. An rfa1-t11 mutation in the ssDNA binding complex RPA partially restores adaptation in rad51Delta mutants and fully restores adaptation in yku70Delta and tid1Delta mutants. Surprisingly, although neither rfa1-t11 nor rad52Delta mutants are adaptation defective, the rad52Delta rfa1-t11 double mutant fails to adapt and exhibits the persistent hyperphosphorylation of the DNA damage checkpoint protein Rad53 after HO induction. We suggest that monitoring of the extent of DNA damage depends on independent binding of RPA and Rad52p to ssDNA, with Rad52p's activity modulated by Rad51p whereas RPA's action depends on Tid1p.     

Separation of phenotypes in mutant alleles of the Schizosaccharomyces pombe cell-cycle checkpoint gene rad1+.

The Schizosaccharomyces pombe rad1+ gene is involved in the G2 DNA damage cell-cycle checkpoint and in coupling mitosis to completed DNA replication. It is also required for viability when the cdc17 (DNA ligase) or wee1 proteins are inactivated. We have introduced mutations into the coding regions of rad1+ by site-directed mutagenesis. The effects of these mutations on the DNA damage and DNA replication checkpoints have been analyzed, as well as their associated phenotypes in a cdc17-K42 or a wee1-50 background. For all alleles, the resistance to radiation or hydroxyurea correlates well with the degree of functioning of checkpoint pathways activated by these treatments. One mutation, rad1-S3, completely abolishes the DNA replication checkpoint while partially retaining the DNA damage checkpoint. As single mutants, the rad1-S1, rad1-S2, rad1-S5, and rad1-S6 alleles have a wild-type phenotype with respect to radiation sensitivity and checkpoint functions; however, like the rad1 null allele, the rad1-S1 and rad1-S2 alleles exhibit synthetic lethality at the restrictive temperature with the cdc17-K42 or the wee1-50 mutation. The rad1-S5 and rad1-S6 alleles allow growth at higher temperatures in a cdc17-K42 or wee1-50 background than does wild-type rad1+, and thus behave like "superalleles." In most cases both chromosomal and multi-copy episomal mutant alleles have been investigated, and the agreement between these two states is very good. We provide evidence that the functions of rad1 can be dissociated into three groups by specific mutations. Models for the action of these rad1 alleles are discussed. In addition, a putative negative regulatory domain of rad1 is identified.     

DDK phosphorylates checkpoint clamp component Rad9 and promotes its release from damaged chromatin

When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1(Cdc7), the Schizosaccharomyces pombe?Dbf4-dependent kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation of Rad9 disrupts its interaction with replication protein A (RPA) and is dependent on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5(TopBP1), and prior phosphorylation by Rad3(ATR). rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and decreased viability after replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to facilitate DNA repair.