HYS2, an essential gene required for DNA replication in Saccharomyces cerevisiae.

To investigate cell cycle regulation at the S or G2 phase in Saccharomyces cerevisiae, we have isolated mutants displaying supersensitivity to hydroxyurea (HU), a chemical that inhibits DNA replication. Such mutants, which we have named hydroxyurea sensitive (hys), defined four linkage groups and we characterized the hys2 mutation in this study. The hys2-1 mutant displays temperature sensitive growth and a constellation of phenotypes indicating defective DNA metabolism. At the restrictive temperature, hys2-1 cells arrest as large budded cells with a single nucleus at the neck of the bud and a short spindle. The hys2-1 mutant exhibits increased rates of chromosome loss and recombination. Additionally, hys2-1 appears to accumulate incompletely replicated DNA that can be detected by a pulse field electrophoresis assay. Finally, deletion of RAD9 in a hys2-1 strain decreases the percentage of arrested cells, suggesting that an intact RAD9-checkpoint is required for the cell cycle arrest in hys2-1 cells. HYS2 encodes a 55 kDa protein that is essential for viability at all temperatures. Taken together, these data suggest that Hys2 plays a role in DNA replication.     

Saccharomyces cerevisiae MGS1 is essential in strains deficient in the RAD6-dependent DNA damage tolerance pathway

Saccharomyces cerevisiae Mgs1 protein, which possesses DNA-dependent ATPase and single strand DNA annealing activities, plays a role in maintaining genomic stability. We found that mgs1 is synthetic lethal with rad6 and exhibits a synergistic growth defect with rad18 and rad5, which are members of the RAD6 epistasis group important for tolerance of DNA damage during DNA replication. The mgs1 mutant is not sensitive to DNA-damaging agents, but the mgs1 rad5 double mutant has increased sensitivity to hydroxyurea and a greatly increased spontaneous mutation rate. Growth defects of mgs1 rad18 double mutants are suppressed by a mutation in SRS2, encoding a DNA helicase, or by overexpression of Rad52. More over, mgs1 mutation suppresses the temperature sensitivity of mutants in POL3, encoding DNA polymerase delta. mgs1 also suppresses the growth defect of a pol3 mutant caused by expression of Escherichia coli RuvC, a bacterial Holliday junction resolvase. These findings suggest that Mgs1 is essential for preventing genome instability caused by replication fork arrest in cells deficient in the RAD6 pathway and may modulate replication fork movement catalyzed by yeast polymerase delta.     

The Large Subunit of Replication Factor C (Rfc1p/Cdc44p) Is Required for DNA Replication and DNA Repair in Saccharomyces Cerevisiae

We used genetic and biochemical techniques to characterize the phenotypes associated with mutations affecting the large subunit of replication factor C (Cdc44p or Rfc1p) in Saccharomyces cerevisiae. We demonstrate that Cdc44p is required for both DNA replication and DNA repair in vivo. Cold-sensitive cdc44 mutants experience a delay in traversing S phase at the restrictive temperature following alpha factor arrest; although mutant cells eventually accumulate with a G2/M DNA content, they undergo a cell cycle arrest and initiate neither mitosis nor a new round of DNA synthesis. cdc44 mutants also exhibit an elevated level of spontaneous mutation, and they are sensitive both to the DNA damaging agent methylmethane sulfonate and to exposure to UV radiation. After exposure to UV radiation, cdc44 mutants at the restrictive temperature contain higher levels of single-stranded DNA breaks than do wild-type cells. This observation is consistent with the hypothesis that Cdc44p is involved in repairing gaps in the DNA after the excision of damaged bases. Thus, Cdc44p plays an important role in both DNA replication and DNA repair in vivo.     

p21CDKN1A does not interfere with loading of PCNA at DNA replication sites, but inhibits subsequent binding of DNA polymerase delta at the G1/S phase transition

The ability of the cyclin-dependent kinase (CDK) inhibitor p21CDKN1A to interact with PCNA recruited to DNA replication sites was investigated to elucidate the relevance of this interaction in cell cycle arrest. To this end, expression of p21 protein fused to green fluorescent protein (GFP) was induced in HeLa cells. G1 phase cell cycle arrest induced by p21GFP occurred also at the G1/S transition, as shown by cyclin A immunostaining of GFP-positive cells. Confocal microscopy analysis and co-immunoprecipitation studies showed that p21GFP co-localized and interacted with chromatin-bound PCNA and CDK2. GFP-p21 mutant forms unable to bind to PCNA (p21PCNA-) or CDK (p21CDK-) induced cell cycle arrest, although immunoprecipitation experiments showed these mutants to be unstable. Expression of HA-tagged p21wt or mutant proteins confirmed the ability of both mutants to arrest cell cycle. p21(wt)HA and p21CDK-HA, but not p21PCNA-, co-localized and co-immunoprecipitated with chromatin-bound PCNA. Association of p21 to chromatin-bound PCNA resulted in the loss of interaction with the p125 catalytic subunit of DNA polymerase delta (pol delta). These results suggest that in vivo p21 does not interfere with loading of PCNA at DNA replication sites, but prevents, or displaces subsequent binding of pol delta to PCNA at the G1/S phase transition.     

Yeast G1 cyclins CLN1 and CLN2 and a GAP-like protein have a role in bud formation.

Cyclin-dependent protein kinases have a central role in cell cycle regulation. In Saccharomyces cerevisiae, Cdc28 kinase and the G1 cyclins Cln1, 2 and 3 are required for DNA replication, duplication of the spindle pole body and bud emergence. These three independent processes occur simultaneously in late G1 when the cells reach a critical size, an event known as Start. At least one of the three Clns is necessary for Start. Cln3 is believed to activate Cln1 and Cln2, which can then stimulate their own accumulation by means of a positive feedback loop. They (or Cln3) also activate another pair of cyclins, Clb5 and 6, involved in initiating S phase. Little is known about the role of Clns in spindle pole body duplication and budding. We report here the isolation of a gene (CLA2/BUD2/ERC25) that codes for a homologue of mammalian Ras-associated GTPase-activating proteins (GAPs) and is necessary for budding only in cln1 cln2 cells. This suggests that Cln1 and Cln2 may have a direct role in bud formation.     

Control of cell division in Saccharomyces cerevisiae mutants defective in adenylate cyclase and cAMP-dependent protein kinase

Examination of the proportion of unbudded cells, terminal nuclear phenotype and DNA content of nuclei indicated that cyr1 mutants of yeast defective in adenylate cyclase activity were arrested at the G1 phase of the cell cycle. The step of G1 arrest due to the cyr1 mutation preceded the step sensitive to the mating pheromone. The temperature-sensitive cyr1 cells did not continue growth, nor retain the capacity to conjugate at a restrictive temperature. The phenotypes of the cyr1 mutant mimicked those of nutritionally limited cells. The G1 arrest caused by the cyr1 mutation was overcome by the presence of a suppressor mutation, bcy1, that resulted in deficiency of a regulatory subunit of cAMP-dependent protein kinase and production of high level of cAMP-independent protein kinase. The bcy1 mutation suppressed G1 arrest caused by nutritional limitation, and continued bud emergence for multiple cycles without further nuclear division. The data suggest that cAMP works as a positive effector at the start of a yeast cell cycle via activation of cAMP-dependent protein kinase.     

Evidence that POB1, a Saccharomyces cerevisiae protein that binds to DNA polymerase alpha, acts in DNA metabolism in vivo.

Potential DNA replication accessory factors from the yeast Saccharomyces cerevisiae have previously been identified by their ability to bind to DNA polymerase alpha protein affinity matrices (J. Miles and T. Formosa, Proc. Natl. Acad. Sci. USA 89:1276-1280, 1992). We have now used genetic methods to characterize the gene encoding one of these DNA polymerase alpha-binding proteins (POB1) to determine whether it plays a role in DNA replication in vivo. We find that yeast cells lacking POB1 are viable but display a constellation of phenotypes indicating defective DNA metabolism. Populations of cells lacking POB1 accumulate abnormally high numbers of enlarged large-budded cells with a single nucleus at the neck of the bud. The average DNA content in a population of cells lacking POB1 is shifted toward the G2 value. These two phenotypes indicate that while the bulk of DNA replication is completed without POB1, mitosis is delayed. Deleting POB1 also causes elevated levels of both chromosome loss and genetic recombination, enhances the temperature sensitivity of cells with mutant DNA polymerase alpha genes, causes increased sensitivity to UV radiation in cells lacking a functional RAD9 checkpoint gene, and causes an increased probability of death in cells carrying a mutation in the MEC1 checkpoint gene. The sequence of the POB1 gene indicates that it is identical to the CTF4 (CHL15) gene identified previously in screens for mutations that diminish the fidelity of chromosome transmission. These phenotypes are consistent with defective DNA metabolism in cells lacking POB1 and strongly suggest that this DNA polymerase alpha-binding protein plays a role in accurately duplicating the genome in vivo.     

Chk1-deficient tumour cells are viable but exhibit multiple checkpoint and survival defects

The conserved protein kinase Chk1 is believed to play an important role in checkpoint responses to aberrant DNA structures; however, genetic analysis of Chk1 functions in metazoans is complicated by lethality of Chk1-deficient embryonic cells. We have used gene targeting to eliminate Chk1 function in somatic DT40 B-lymphoma cells. We find that Chk1-deficient DT40 cells are viable, but fail to arrest in G(2)/M in response to and are hypersensitive to killing by ionizing radiation. Chk1-deficient cells also fail to maintain viable replication forks or suppress futile origin firing when DNA polymerase is inhibited, leading to incomplete genome duplication and diminished cell survival after release from replication arrest. In contrast to embryonic cells, however, Chk1 is not required to delay mitosis when DNA synthesis is inhibited. Thus, Chk1 is dispensable for normal cell division in somatic DT40 cells but is essential for DNA damage-induced G(2)/M arrest and a subset of replication checkpoint responses. Furthermore, Chk1-dependent processes promote tumour cell survival after perturbations of DNA structure or metabolism.     

Saccharomyces cerevisiae lacking Snm1, Rev3 or Rad51 have a normal S-phase but arrest permanently in G2 after cisplatin treatment

The role of Snm1, Rev3 and Rad51 in S-phase after cisplatin (CDDP) DNA treatment has been examined. When isogenic deletion mutants snm1 delta, rev3 delta and rad51 delta were arrested in G1 and treated with doses of CDDP causing significant lethality (<20% survival in the mutant strains), they progressed through S-phase with normal kinetics. The mutants arrested in G2 like wild-type cells, however they did not exit the arrest and reenter the cell cycle. This finding demonstrates that these genes are not required to allow DNA replication in the presence of damage. Therefore, Snm1, Rev3 and Rad51 may act after S to allow repair. At high levels of damage (<40% survival in wild-type cells) S-phase was slowed in a MEC1-dependent fashion. The cross-link incision kinetics of snm1 delta and rev3 delta mutants were also examined; both showed no deficiencies in incision of cross-linked DNA.     

Rhp51-dependent recombination intermediates that do not generate checkpoint signal are accumulated in Schizosaccharomyces pombe rad60 and smc5/6 mutants after release from replication arrest

The Schizosaccharomyces pombe rad60 gene is essential for cell growth and is involved in repairing DNA double-strand breaks. Rad60 physically interacts with and is functionally related to the structural maintenance of chromosomes 5 and 6 (SMC5/6) protein complex. In this study, we investigated the role of Rad60 in the recovery from the arrest of DNA replication induced by hydroxyurea (HU). rad60-1 mutant cells arrested mitosis normally when treated with HU. Significantly, Rad60 function is not required during HU arrest but is required on release. However, the mutant cells underwent aberrant mitosis accompanied by irregular segregation of chromosomes, and DNA replication was not completed, as revealed by pulsed-field gel electrophoresis. The deletion of rhp51 suppressed the aberrant mitosis of rad60-1 cells and caused mitotic arrest. These results suggest that Rhp51 and Rad60 are required for the restoration of a stalled or collapsed replication fork after release from the arrest of DNA replication by HU. The rad60-1 mutant was proficient in Rhp51 focus formation after release from the HU-induced arrest of DNA replication or DNA-damaging treatment. Furthermore, the lethality of a rad60-1 rqh1Delta double mutant was suppressed by the deletion of rhp51 or rhp57. These results suggest that Rad60 is required for recombination repair at a step downstream of Rhp51. We propose that Rhp51-dependent DNA structures that cannot activate the mitotic checkpoints accumulate in rad60-1 cells.     

The ts41 mutation in Chinese hamster cells leads to successive S phases in the absence of intervening G2, M, and G1.

The ts41 mutation of Chinese hamster cells was first isolated and characterized by Hirschberg and Marcus (1982) who showed that at nonpermissive temperature, cells accumulate up to 16C equivalents of DNA. Here we show that the mutation is recessive and at nonpermissive temperature, cells replicate their genome normally, but instead of going on into G2, M, and G1, they pass directly into a second S phase. Entry into a second S phase does not require serum nor is it inhibited by G2 checkpoints or mitotic inhibitors. Temperature-shift experiments suggest that the ts41 gene product participates in two functions in the cell cycle: entry into mitosis and inhibition of entry into S phase. The ts41 mutation seems to define a class of cell cycle mutant that couples the sequential events of DNA replication and mitosis.     

Status of calcium influx in cell cycle of S. cerevisiae

The transport of calcium was assayed in exponentially growing and G1 arrested temperature sensitive cdc mutants of Saccharomyces cerevisiae. There was no statistically significant difference in the rate of Ca2+ influx in cdc 28, cdc 37 and cdc 4 arrested cells, as well as in wild type cells arrested in G1 phase in comparison to exponentially growing cells. There was however a significant increase in Ca2+ uptake in cdc 7 and cdc 24 arrested cells. The former is known to arrest before bud emergence and initiation of DNA synthesis; arrest of the latter affects bud formation while DNA synthesis continues. The results suggest that Ca2+ may have a role in bud formation and growth.     

Mitotic UV Irradiation Induces a DNA Replication-Licensing Defect that Potentiates G1 Arrest Response

Cdt1 begins to accumulate in M phase and has a key role in establishing replication licensing at the end of mitosis or in early G1 phase. Treatments that damage the DNA of cells, such as UV irradiation, induce Cdt1 degradation through PCNA-dependent CRL4-Cdt2 ubiquitin ligase. How Cdt1 degradation is linked to cell cycle progression, however, remains unclear. In G1 phase, when licensing is established, UV irradiation leads to Cdt1 degradation, but has little effect on the licensing state. In M phase, however, UV irradiation does not induce Cdt1 degradation. When mitotic UV-irradiated cells were released into G1 phase, Cdt1 was degraded before licensing was established. Thus, these cells exhibited both defective licensing and G1 cell cycle arrest. The frequency of G1 arrest increased in cells expressing extra copies of Cdt2, and thus in cells in which Cdt1 degradation was enhanced, whereas the frequency of G1 arrest was reduced in cell expressing an extra copy of Cdt1. The G1 arrest response of cells irradiated in mitosis was important for cell survival by preventing the induction of apoptosis. Based on these observations, we propose that mammalian cells have a DNA replication-licensing checkpoint response to DNA damage induced during mitosis.     

The SLT2 (MPK1) MAP kinase homolog is involved in polarized cell growth in Saccharomyces cerevisiae

Bud emergence, spindle pole body duplication and DNA replication are all dependent on the activation of the CDC28 protein kinase at the Start point in the G1 phase of the cell cycle. Bud emergence requires polarization of the cytoskeleton and secretory vesicles to a specific site on the cell surface. Cdc28p activated by G1-cyclins triggers polarization of actin to the site of bud emergence and favors apical bud growth (Lew, D. J., and S. I. Reed. 1993. J. Cell Biol. 120:1305- 1320). We isolated slt2-1 as a mutation that enhances the division defect of cdc28 mutants with defects at Start. Slt2p(Mpk1p) is a member of the MAP kinase family (Lee, K. S., K. Irie, Y. Gotoh, Y. Watanabe, H. Araki, E. Nishida, K. Matsumoto, and D. E. Levin. 1993. Mol. Cell. Biol. 13:3067-3075). We show that slt2 mutants exhibit phenotypes similar to those shown by mutants of the yeast actin cytoskeleton, including delocalization of chitin deposition and of actin cortical spots and the accumulation of secretory pathway membranes and vesicles. Furthermore, slt2::HIS3 act1-1 and slt2::HIS3 myo2-66 double mutants are inviable. We suggest that Slt2p functions downstream or in parallel with Cdc28p in promoting bud formation and apical growth.     

Functional interaction between the PKC1 pathway and CDC31 network of SPB duplication genes

The earliest known step in yeast spindle pole body (SPB) duplication requires Cdc31p and Kar1p, two physically interacting SPB components, and Dsk2p and Rad23p, a pair of ubiquitin-like proteins. Components of the PKC1 pathway were found to interact with these SPB duplication genes in two independent genetic screens. Initially, SLG1 and PKC1 were obtained as high-copy suppressors of dsk2Delta rad23Delta and a mutation in MPK1 was synthetically lethal with kar1-Delta17. Subsequently, we demonstrated extensive genetic interactions between the PKC1 pathway and the SPB duplication mutants that affect Cdc31p function. The genetic interactions are unlikely to be related to the cell-wall integrity function of the PKC1 pathway because the SPB mutants did not exhibit cell-wall defects. Overexpression of multiple PKC1 pathway components suppressed the G2/M arrest of the SPB duplication mutants and mutations in MPK1 exacerbated the cell cycle arrest of kar1-Delta17, suggesting a role for the PKC1 pathway in SPB duplication. We also found that mutations in SPC110, which encodes a major SPB component, showed genetic interactions with both CDC31 and the PKC1 pathway. In support of the model that the PKC1 pathway regulates SPB duplication, one of the phosphorylated forms of Spc110p was absent in pkc1 and mpk1Delta mutants.     

A temperature-sensitive mutation in the WD repeat-containing protein Smu1 is related to maintenance of chromosome integrity

Temperature-sensitive CHO-K1 mutant cell line tsTM18 exhibits chromosomal instability and cell cycle arrest at S and G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39 degrees C. To identify the causative mutation, we fused tsTM18 cells with normal human cells to generate hybrids carrying fragments of human chromosomes. Analysis of chromosome content of temperature-resistant transformants and introduction of a bacterial artificial chromosome containing part of human chromosome 9 led to isolation of the human SMU1 gene. Comparison of sequences of the Smu1 gene from wild-type and mutant cells revealed that the mutant phenotype is caused by a G-to-A transition that yields a gly-to-arg substitution at position 489 in hamster Smu1. The substituted glycine is located in the WD-repeat domain of Smu1. Single-stranded DNA accumulated in the nuclei of mutant cells at 39 degrees C. Furthermore, cdc2 kinase was not activated during G2 phase, and there was no chromosome segregation due to incomplete assembly of the spindle during M phase. Thus, Smu1 appears to be involved directly or indirectly in DNA replication, activation of cdc2 kinase, spindle assembly, and maintenance of chromosome integrity, reflecting the important roles of Smu1 in cellular function.     

Sap1 promotes the association of the replication fork protection complex with chromatin and is involved in the replication checkpoint in Schizosaccharomyces pombe

Sap1 is involved in replication fork pausing at rDNA repeats and functions during mating-type switching in Schizosaccharomyces pombe. These two roles are dependent on the ability of Sap1 to bind specific DNA sequences at the rDNA and mating-type loci, respectively. In S. pombe, Swi1 and Swi3 form the replication fork protection complex (FPC) and play important roles in the activation of the replication checkpoint and the stabilization of stalled replication forks. Here we describe the roles of Sap1 in the replication checkpoint. We show that Sap1 is involved in the activation of the replication checkpoint kinase Cds1 and that sap1 mutant cells accumulate spontaneous DNA damage during the S- and G2-phases, which is indicative of fork damage. We also show that sap1 mutants have a defect in the resumption of DNA replication after fork arrest. Sap1 is localized at the replication origin ori2004 and this localization is required for the association of the FPC with chromatin. We propose that Sap1 is required to recruit the FPC to chromatin, thereby contributing to the activation of the replication checkpoint and the stabilization of replication forks.