G2 / M checkpoint genes of Saccharomyces cerevisiae : further evidence for roles in DNA replication and / or repair

We have cloned, sequenced and disrupted the checkpoint genes RAD17, RAD24 and MEC3 of Saccharomyces cerevisiae. Mec3p shows no strong similarity to other proteins currently in the database. Rad17p is similar to Rec1 from Ustilago maydis, a 3′ to 5′ DNA exonuclease/checkpoint protein, and the checkpoint protein Rad1p from Schizosaccharomyces pombe (as we previously reported). Rad24p shows sequence similarity to replication factor C (RFC) subunits, and the S. pombe Rad17p checkpoint protein, suggesting it has a role in DNA replication and/or repair. This hypothesis is supported by our genetic experiments which show that overexpression of RAD24 strongly reduces the growth rate of yeast strains that are defective in the DNA replication/repair proteins Rfc1p (cdc44), DNA polα (cdc17) and DNA polδ (cdc2) but has much weaker effects on cdc6, cdc9, cdc15 and CDC ⁺ strains. The idea that RAD24 overexpression induces DNA damage, perhaps by interfering with replication/repair complexes, is further supported by our observation that RAD24 overexpression increases mitotic chromosome recombination in CDC ⁺ strains. Although RAD17, RAD24 and MEC3 are not required for cell cycle arrest when S phase is inhibited by hydroxyurea (HU), they do contribute to the viability of yeast cells grown in the presence of HU, possibly because they are required for the repair of HU-induced DNA damage. In addition, all three are required for the rapid death of cdc13 rad9 mutants. All our data are consistent with models in which RAD17, RAD24 and MEC3 are coordinately required for the activity of one or more DNA repair pathways that link DNA damage to cell cycle arrest. Received: 8 April 1997 / Accepted: 10 May 1997     

Role of a Complex Containing Rad17, Mec3, and Ddc1 in the Yeast DNA Damage Checkpoint Pathway

Genetic analysis has suggested that RAD17, RAD24, MEC3, and DDC1 play similar roles in the DNA damage checkpoint control in budding yeast. These genes are required for DNA damage-induced Rad53 phosphorylation and considered to function upstream of RAD53 in the DNA damage checkpoint pathway. Here we identify Mec3 as a protein that associates with Rad17 in a two-hybrid screen and demonstrate that Rad17 and Mec3 interact physically in vivo. The amino terminus of Rad17 is required for its interaction with Mec3, and the protein encoded by the rad17-1 allele, containing a missense mutation at the amino terminus, is defective for its interaction with Mec3 in vivo. Ddc1 interacts physically and cosediments with both Rad17 and Mec3, indicating that these three proteins form a complex. On the other hand, Rad24 is not found to associate with Rad17, Mec3, and Ddc1. DDC1 overexpression can partially suppress the phenotypes of the rad24Δ mutation: sensitivity to DNA damage, defect in the DNA damage checkpoint and decrease in DNA damage-induced phosphorylation of Rad53. Taken together, our results suggest that Rad17, Mec3, and Ddc1 form a complex which functions downstream of Rad24 in the DNA damage checkpoint pathway.     

Cdc20, a β-transducin homologue,links RAD9-mediated G2/M checkpoint control to mitosis in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, the DNA damage-induced G2 arrest requires the checkpoint control genes RAD9, RAD17, RAD24, MEC1, MEC2 and MEC3. These genes also prevent entry into mitosis of a temperature-sensitive mutant, cdc13, that accumulates chromosome damage at 37^°?C. Here we show that a cdc13 mutant overexpressing Cdc20, a β-transducin homologue, no longer arrests in G2 at the restrictive temperature but instead undergoes nuclear division, exits mitosis and enters a subsequent division cycle, which suggests that the DNA damage-induced G2/M checkpoint control is not functional in these cells. This is consistent with our observation that overexpression of CDC20 in wild-type cells results in increased sensitivity to UV irradiation. Overproduction of Cdc20 does not influence the arrest phenotype of the cdc mutants whose cell cycle block is independent of RAD9-mediated checkpoint control. Therefore, we suggest that the DNA damage-induced checkpoint controls prevent mitosis by inhibiting the nuclear division pathway requiring CDC20 function.     

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.     

Human and Mouse Homologs of theSchizosaccharomyces pombe rad17+Cell Cycle Checkpoint Control Gene

TheSchizosaccharomyces pombe rad17⁺cell cycle checkpoint control gene is required for S-phase and G2/M arrest in response to both DNA damage and incomplete DNA replication. We isolated and characterized the putative human (RAD17Sp) and mouse (mRAD17Sp) homologs of theS. pombeRad17 (Rad17Sp) protein. The humanRAD17Spopen reading frame (ORF) encodes a protein of 681 amino acids; themRAD17SpORF codes for a protein of 688 amino acids. ThemRAD17Spmessenger is highly expressed in the testis as a single 3-kb mRNA species. The human RAD17Sp and mRAD17Sp proteins are 24% identical and 46% similar to theS.pombeRad17Sp protein. Sequence homology was also noted with theSaccharomyces cerevisiaeRad24Sc (which is the structural counterpart ofS.pombeRad17Sp) and structurally related polypeptides fromCaenorhabditis elegans, Arabidopsis thaliana, Pyrococcus horikoshii,andDrosophila melanogaster.The degree of conservation between the mammalian RAD17Sp proteins and those of the other species is consistent with the evolutionary distance between the species, indicating that these proteins are most likely true counterparts. In addition, homology was found between the Rad17Sp homologs and proteins identified as components of mammalian replication factor C (RF-C)/activator 1, especially in several highly conserved RF-C-like domains including a “Walker A” motif. Using FISH and analysis of a panel of rodent–human cell hybrids, the humanRAD17Spgene (HGMW-approved symbolRAD17could be localized on human chromosome 5q13–q14, a region implicated in the etiology of small cell lung carcinoma, non-small-cell lung carcinoma, duodenal adenocarcinoma, and head and neck squamous cell carcinoma. Our results suggest that the structure and function of the checkpoint “rad” genes in the G2/M checkpoint pathway are evolutionary conserved between yeast and higher eukaryotes.     

Function of Rad17/Mec3/Ddc1 and its partial complexes in the DNA damage checkpoint

The Saccharomyces cerevisiae heterotrimeric checkpoint clamp consisting of the Rad17, Mec3, and Ddc1 subunits (Rad17/3/1, the 9-1-1 complex in humans) is an early response factor to DNA damage in a signal transduction pathway leading to the activation of the checkpoint system and eventually to cell cycle arrest. These subunits show structural similarities with the replication clamp PCNA and indeed, it was demonstrated in vitro that Rad17/3/1 could be loaded onto DNA by checkpoint specific clamp loader Rad24-RFC, analogous to the PCNA-RFC clamp-clamp loader system. We have studied the interactions between the checkpoint clamp subunits and the activity of partial clamp complexes. We find that none of the possible partial complexes makes up a clamp that can be loaded onto DNA by Rad24-RFC. In agreement, overexpression of DDC1 or RAD17 in a MEC3Delta strain, or of MEC3 or RAD17 in a DDC1Delta strain shows no rescue of damage sensitivity.     

Mec1p is essential for phosphorylation of the yeast DNA damage checkpoint protein Ddc1p, which physically interacts with Mec3p.

Checkpoints prevent DNA replication or nuclear division when chromosomes are damaged. The Saccharomyces cerevisiae DDC1 gene belongs to the RAD17, MEC3 and RAD24 epistasis group which, together with RAD9, is proposed to act at the beginning of the DNA damage checkpoint pathway. Ddc1p is periodically phosphorylated during unperturbed cell cycle and hyperphosphorylated in response to DNA damage. We demonstrate that Ddc1p interacts physically in vivo with Mec3p, and this interaction requires Rad17p. We also show that phosphorylation of Ddc1p depends on the key checkpoint protein Mec1p and also on Rad24p, Rad17p and Mec3p. This suggests that Mec1p might act together with the Rad24 group of proteins at an early step of the DNA damage checkpoint response. On the other hand, Ddc1p phosphorylation is independent of Rad53p and Rad9p. Moreover, while Ddc1p is required for Rad53p phosphorylation, it does not play any major role in the phosphorylation of the anaphase inhibitor Pds1p, which requires RAD9 and MEC1. We suggest that Rad9p and Ddc1p might function in separated branches of the DNA damage checkpoint pathway, playing different roles in determining Mec1p activity and/or substrate specificity.     

RAD53 is limiting in double-strand break repair and in protection against toxicity associated with ribonucleotide reductase inhibition

The yeast Chk2/Chk1 homolog Rad53 is a central component of the DNA damage checkpoint system. While it controls genotoxic stress responses such as cell cycle arrest, replication fork stabilization and increase in dNTP pools, little is known about the consequences of reduced Rad53 levels on the various cellular endpoints or about its roles in dealing with chronic vs. acute genotoxic challenges. Using a tetraploid gene dosage model in which only one copy of the yeast RAD53 is functional (simplex), we found that the simplex strain was not sensitive to acute UV radiation or chronic MMS exposure. However, the simplex strain was sensitized to chronic exposure of the ribonucleotide reductase inhibitor hydroxyurea (HU). Surprisingly, reduced RAD53 gene dosage did not affect sensitivity to HU acute exposure, indicating that immediate checkpoint responses and recovery from HU-induced stress were not compromised. Interestingly, cells of most of the colonies that arise after chronic HU exposure acquired heritable resistance to HU. We also found that short HU exposure before and after treatment of G₂ cells with ionizing radiation (IR) reduced the capability of RAD53 simplex cells to repair DSBs, in agreement with sensitivity of RAD53 simplex strain to high doses of IR. We propose that a modest reduction in Rad53 activity can impact the activation of the ribonucleotide reductase catalytic subunit Rnr1 following stress, reducing the ability to generate nucleotide pools sufficient for DNA repair and replication. At the same time, reduced Rad53 activity may lead to genome instability and to the acquisition of drug resistance before and/or during the chronic exposure to HU. These results have implications for developing drug enhancers as well as for understanding mechanisms of drug resistance in cells compromised for DNA damage checkpoint.     

Dual cell cycle checkpoints sensitive to chromosome replication and DNA damage in the budding yeast Saccharomyces cerevisiae.

In eucaryotic cells chromosomes must be fully replicated and repaired before mitosis begins. Genetic studies indicate that this dependence of mitosis on completion of DNA replication and DNA repair derives from a negative control called a checkpoint which somehow checks for replication and DNA damage and blocks cell entry into mitosis. Here we summarize our current understanding of the genetic components of the cell cycle checkpoint in budding yeast. Mutants were identified and their phase and signal specificity tested primarily through interactions of the arrest-defective mutants with cell division cycle mutants. The results indicate that dual checkpoint controls exist in budding yeast, one control sensitive to inhibition of DNA replication (S-phase checkpoint), and a distinct but overlapping control sensitive to DNA repair (G2 checkpoint). Six genes are required for arrest in G2 phase after DNA damage (RAD9, RAD17, RAD24, MEC1, MEC2, and MEC3), and two of these are also essential for arrest in S phase when DNA replication is blocked (MEC1 and MEC2).     

cDNA Cloning and Gene Mapping of Human Homologs forSchizosaccharomyces pombe rad17, rad1,andhus1and Cloning of Homologs from Mouse,Caenorhabditis elegans,andDrosophila melanogaster

Mutations in DNA repair/cell cycle checkpoint genes can lead to the development of cancer. The cloning of human homologs of yeast DNA repair/cell cycle checkpoint genes should yield candidates for human tumor suppressor genes as well as identifying potential targets for cancer therapy. TheSchizosaccharomyces pombegenesrad17, rad1,andhus1have been identified as playing roles in DNA repair and cell cycle checkpoint control pathways. We have cloned the cDNA for the human homolog ofS. pombe rad17,RAD17, which localizes to chromosomal location 5q13 by fluorescencein situhybridization and radiation hybrid mapping; the cDNA for the human homolog ofS. pombe rad1,RAD1, which maps to 5p14–p13.2; and the cDNA for the human homolog ofS. pombe hus1,HUS1, which maps to 7p13–p12. The human gene loci have previously been identified as regions containing tumor suppressor genes. In addition, we report the cloning of the cDNAs for genes related toS. pombe rad17, rad9, rad1,andhus1from mouse,Caenorhabditis elegans,andDrosophila melanogaster.These includeRad17andRad9fromD. melanogaster,hpr-17 and hpr-1 fromC. elegans,and RAD1 and HUS1 from mouse. The identification of homologs of theS. pomberad checkpoint genes from mammals, arthropods, and nematodes indicates that this cell cycle checkpoint pathway is conserved throughout eukaryotes.     

Cdc5 blocks in vivo rad53 activity,but not in situ activity (ISA)

DNA damage promotes the activation of a signal transduction cascade referred to as the DNA damage checkpoint. This pathway initiates with the Mec1/ATR kinase, which then phosphorylates the Rad53/Chk2 kinase. Mec1 phosphorylation of Rad53 is then thought to promote Rad53 autophosphorylation, ultimately leading to a fully active Rad53 molecule that can go on to phosphorylate substrates important for DNA damage resistance. In the absence of DNA repair, this checkpoint is eventually downregulated in a Cdc5-dependent process referred to as checkpoint adaptation. Recently, we showed that overexpression of Cdc5 leads to checkpoint inactivation and loss of the strong electrophoretic shift associated with Rad53 inactivation. Interestingly, this same overexpression did not strongly inhibit Rad53 autophosphorylation activity as measured by the in situ assay (ISA). The ISA involves incubating the re-natured Rad53 protein with γ ³²P labeled ATP after electrophoresis and western blotting. Using a newly identified Rad53 target, we show that despite strong ISA activity, Rad53 does not maintain phosphorylation of this substrate. We hypothesize that, during adaptation, Rad53 may be in a unique state in which it maintains some Mec1 phosphorylation but does not have the auto-phosphorylations required for full activity towards exogenous substrates.Key words: DNA damage, checkpoint, adaptation, CDC5, RAD53, ISA     

Critical Functions of Rpa3/Ssb3 in S-Phase DNA Damage Responses in Fission Yeast

Replication Protein A (RPA) is a heterotrimeric, single-stranded DNA (ssDNA)–binding complex required for DNA replication and repair, homologous recombination, DNA damage checkpoint signaling, and telomere maintenance. Whilst the larger RPA subunits, Rpa1 and Rpa2, have essential interactions with ssDNA, the molecular functions of the smallest subunit Rpa3 are unknown. Here, we investigate the Rpa3 ortholog Ssb3 in Schizosaccharomyces pombe and find that it is dispensable for cell viability, checkpoint signaling, RPA foci formation, and meiosis. However, increased spontaneous Rad11^Rpa1 and Rad22^Rad52 nuclear foci in ssb3Δ cells indicate genome maintenance defects. Moreover, Ssb3 is required for resistance to genotoxins that disrupt DNA replication. Genetic interaction studies indicate that Ssb3 has a close functional relationship with the Mms1-Mms22 protein complex, which is required for survival after DNA damage in S-phase, and with the mitotic functions of Mus81-Eme1 Holliday junction resolvase that is required for recovery from replication fork collapse. From these studies we propose that Ssb3 plays a critical role in mediating RPA functions that are required for repair or tolerance of DNA lesions in S-phase. Rpa3 orthologs in humans and other species may have a similar function.     

The fission yeast pfh1(+) gene encodes an essential 5' to 3' DNA helicase required for the completion of S-phase

The Cdc24 protein plays an essential role in chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe, most likely via its direct interaction with Dna2, a conserved endonuclease–helicase protein required for Okazaki fragment processing. To gain insights into Cdc24 function, we isolated cold-sensitive chromosomal suppressors of the temperature-sensitive cdc24-M38 allele. One of the complementation groups of such suppressors defined a novel gene, pfh1⁺, encoding an 805 amino acid nuclear protein highly homologous to the Saccharomyces cerevisiae Pif1p and Rrm3p DNA helicase family proteins. The purified Pfh1 protein displayed single-stranded DNA-dependent ATPase activity as well as 5′ to 3′ DNA helicase activity in vitro. Reverse genetic analysis in S.pombe showed that helicase activity was essential for the function of the Pfh1 protein in vivo. Schizosaccharomyces pombe cells carrying the cold-sensitive pfh1-R20 allele underwent cell cycle arrest in late S/G2-phase of the cell cycle when shifted to the restrictive temperature. This arrest was dependent upon the presence of a functional late S/G2 DNA damage checkpoint, suggesting that Pfh1 is required for the comple tion of DNA replication. Furthermore, at their permissive temperature pfh1-R20 cells were highly sensitive to the DNA-alkylating agent methyl methanesulphonate, implying a further role for Pfh1 in the repair of DNA damage.     

Accumulation of sumoylated Rad52 in checkpoint mutants perturbed in DNA replication

Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA-damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, chromatin immunoprecipitation (ChIP) on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double mutation of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD53 single mutation. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed.     

Unligated Okazaki Fragments Induce PCNA Ubiquitination and a Requirement for Rad59-Dependent Replication Fork Progression

Deficiency in DNA ligase I, encoded by CDC9 in budding yeast, leads to the accumulation of unligated Okazaki fragments and triggers PCNA ubiquitination at a non-canonical lysine residue. This signal is crucial to activate the S phase checkpoint, which promotes cell cycle delay. We report here that a pol30-K107 mutation alleviated cell cycle delay in cdc9 mutants, consistent with the idea that the modification of PCNA at K107 affects the rate of DNA synthesis at replication forks. To determine whether PCNA ubiquitination occurred in response to nicks or was triggered by the lack of PCNA-DNA ligase interaction, we complemented cdc9 cells with either wild-type DNA ligase I or a mutant form, which fails to interact with PCNA. Both enzymes reversed PCNA ubiquitination, arguing that the modification is likely an integral part of a novel nick-sensory mechanism and not due to non-specific secondary mutations that could have occurred spontaneously in cdc9 mutants. To further understand how cells cope with the accumulation of nicks during DNA replication, we utilized cdc9-1 in a genome-wide synthetic lethality screen, which identified RAD59 as a strong negative interactor. In comparison to cdc9 single mutants, cdc9 rad59Δ double mutants did not alter PCNA ubiquitination but enhanced phosphorylation of the mediator of the replication checkpoint, Mrc1. Since Mrc1 resides at the replication fork and is phosphorylated in response to fork stalling, these results indicate that Rad59 alleviates nick-induced replication fork slowdown. Thus, we propose that Rad59 promotes fork progression when Okazaki fragment processing is compromised and counteracts PCNA-K107 mediated cell cycle arrest.     

Hsk1 kinase and Cdc45 regulate replication stress-induced checkpoint responses in fission yeast

In fission yeast, replication fork arrest activates the replication checkpoint effector kinase Cds1^Chk2/Rad53 through the Rad3^ATR/Mec1-Mrc1^Claspin pathway. Hsk1, the Cdc7 homologue of fission yeast required for efficient initiation of DNA replication, is also required for Cds1 activation. Hsk1 kinase activity is required for induction and maintenance of Mrc1 hyperphosphorylation, which is induced by replication fork block and mediated by Rad3. Rad3 kinase activity does not change in an hsk1 temperature-sensitive mutant, and Hsk1 kinase activity is not affected by rad3 mutation. Hsk1 kinase vigorously phosphorylates Mrc1 in vitro, predominantly at non-SQ/TQ sites, but this phosphorylation does not seem to affect the Rad3 action on Mrc1. Interestingly, the replication stress-induced activation of Cds1 and hyperphosphorylation of Mrc1 is almost completely abrogated in an initiation-defective mutant of cdc45, but not in an mcm2 or polε mutant. The results suggest that Hsk1-mediated loading of Cdc45 onto replication origins may play important roles in replication stress-induced checkpoint.