CDC45, a novel yeast gene that functions with the origin recognition complex and Mcm proteins in initiation of DNA replication.

The CDC45 gene of Saccharomyces cerevisiae was isolated by complementation of the cold-sensitive cdc45-1 mutant and shown to be essential for cell viability. Although CDC45 genetically interacts with a group of MCM genes (CDC46, CDC47, and CDC54), the predicted sequence of its protein product reveals no significant sequence similarity to any known Mcm family member. Further genetic characterization of the cdc45-1 mutant demonstrated that it is synthetically lethal with orc2-1, mcm2-1, and mcm3-1. These results not only reveal a functional connection between the origin recognition complex (ORC) and Cdc45p but also extend the CDC45-MCM genetic interaction to all known MCM family members that were shown to be involved in replication initiation. Initiation of DNA replication in cdc45-1 cells was defective, causing a delayed entry into S phase at the nonpermissive temperature, as well as a high plasmid loss rate which could be suppressed by tandem copies of replication origins. Furthermore, two-dimensional gels directly showed that chromosomal origins fired less frequently in cdc45-1 cells at the nonpermissive temperature. These findings suggest that Cdc45p, ORC, and Mcm proteins act in concert for replication initiation throughout the genome.     

Schizosaccharomyces pombe Mcm3p, an essential nuclear protein, associates tightly with Nda4p (Mcm5p).

MCM proteins are required for the proper regulation of DNA replication. There are six MCM proteins in all eukaryotes which interact to form a large complex. We report the cloning of fission yeast mcm3 +. mcm3 + is essential and spores carrying a Delta mcm3 disruption arrest with an apparently replicated DNA content. The protein is found constitutively in the nucleus and levels remain constant throughout the cell cycle. Mcm3p binds particularly tightly to Nda4p (Mcm5p), but is loosely associated with the other Schizosaccharomyces pombe MCM proteins. Thus, Mcm3p is a peripheral MCM subunit.     

Purification and functional inactivation of the fission yeast MCM(MCM-BP) complex

The MCM (mini-chromosome maintenance) complex is the core of the eukaryotic replicative helicase and comprises six proteins, Mcm2-Mcm7. In humans, a variant form of the complex has Mcm2 replaced by the MCM-BP protein. Recent results suggest that a similar complex exists in fission yeast with an essential role in DNA replication and cell cycle progression. Here, we describe the purification and subunit composition of the fission yeast MCM(Mcb1) complex. Using newly generated temperature-sensitive alleles, we show that loss of MCM(Mcb1) function leads to accumulation of DNA damage, checkpoint activation and cell cycle arrest, and provide evidence for a role for MCM(Mcb1) in meiosis.     

A yeast model for the study of human DFNA5, a gene mutated in nonsyndromic hearing impairment

A mutation in human DFNA5 is associated with autosomal dominant nonsyndromic hearing impairment. The function of DFNA5 protein remains unknown and no experimental model has been described so far. Here we describe fission yeast Schizosaccharomyces pombe as a model organism for studying the function of heterologously expressed DFNA5. We have expressed wild-type as well as mutant DFNA5 alleles under control of regulatable nmt1 promoter. Yeast cells tolerated expression of wild-type DFNA5, while expression of the mutant DFNA5 allele, which is responsible for nonsyndromic autosomal dominant hearing impairment, led to cell cycle arrest. We identified new rat and horse DFNA5 homologues and we describe a domain of homology shared between DFNA5 and the Mcm10 family of DNA replication proteins. Genetic interactions between heterologously expressed DFNA5 and a fission yeast cdc23 (mcm10) mutant support a possible link between DFNA5 and Mcm10 proteins.     

Functional conservation of beta-hairpin DNA binding domains in the Mcm protein of Methanobacterium thermoautotrophicum and the Mcm5 protein of Saccharomyces cerevisiae

Mcm proteins are an important family of evolutionarily conserved helicases required for DNA replication in eukaryotes. The eukaryotic Mcm complex consists of six paralogs that form a heterohexameric ring. Because the intact Mcm2-7 hexamer is inactive in vitro, it has been difficult to determine the precise function of the different subunits. The solved atomic structure of an archaeal minichromosome maintenance (MCM) homolog provides insight into the function of eukaryotic Mcm proteins. The N-terminal positively charged central channel in the archaeal molecule consists of beta-hairpin domains essential for DNA binding in vitro. Eukaryotic Mcm proteins also have beta-hairpin domains, but their function is unknown. With the archaeal atomic structure as a guide, yeast molecular genetics was used to query the function of the beta-hairpin domains in vivo. A yeast mcm5 mutant with beta-hairpin mutations displays defects in the G1/S transition of the cell cycle, the initiation phase of DNA replication, and in the binding of the entire Mcm2-7 complex to replication origins. A similar mcm4 mutation is synthetically lethal with the mcm5 mutation. Therefore, in addition to its known regulatory role, Mcm5 protein has a positive role in origin binding, which requires coordination by all six Mcm2-7 subunits in the hexamer.     

Role of DNA replication proteins in double-strand break-induced recombination in Saccharomyces cerevisiae

Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4 Delta strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase alpha (Pol alpha), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G(2)-arrested cells. Whereas PCNA was still essential for MAT switching, neither Pol alpha nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Pol alpha-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.     

Minichromosome maintenance as a genetic assay for defects in DNA replication.

Minichromosome maintenance (mcm) is an effective genetic assay for mutants defective in DNA replication. Two classes of mcm mutants have been identified using this screen: those that differentially affect the activities of certain autonomously replicating sequences (ARSs) and those that uniformly affect the activities of all ARSs. The ARS-specific MCM genes are essential for the initiation of DNA replication. Among these are members of the MCM2-7 family that encode subunits of the preinitiation complex and MCM10, whose gene product interacts with members of the Mcm2-7 proteins. Among the ARS-nonspecific MCM gene products are chromosome transmission factors. Refinement of this genetic assay as a screening tool and further analysis of existing mcm mutants may reveal new replication initiation proteins.     

Schizosaccharomyces pombe minichromosome maintenance-binding protein (MCM-BP) antagonizes MCM helicase

The minichromosome maintenance (MCM) complex, a replicative helicase, is a heterohexamer essential for DNA duplication and genome stability. We identified Schizosaccharomyces pombe mcb1(+) (Mcm-binding protein 1), an apparent orthologue of the human MCM-binding protein that associates with a subset of MCM complex proteins. mcb1(+) is an essential gene. Deletion of mcb1(+) caused cell cycle arrest after several generations with a cdc phenotype and disrupted nuclear structure. Mcb1 is an abundant protein, constitutively present across the cell cycle. It is widely distributed in cytoplasm and nucleoplasm and bound to chromatin. Co-immunoprecipitation suggested that Mcb1 interacts robustly with Mcm3-7 but not Mcm2. Overproduction of Mcb1 disrupted the association of Mcm2 with other MCM proteins, resulting in inhibition of DNA replication, DNA damage, and activation of the checkpoint kinase Chk1. Thus, Mcb1 appears to antagonize the function of MCM helicase.     

Schizosaccharomyces pombe Hsk1p is a potential cds1p target required for genome integrity

The fission yeast Hsk1p kinase is an essential activator of DNA replication. Here we report the isolation and characterization of a novel mutant allele of the gene. Consistent with its role in the initiation of DNA synthesis, hsk1(ts) genetically interacts with several S-phase mutants. At the restrictive temperature, hsk1(ts) cells suffer abnormal S phase and loss of nuclear integrity and are sensitive to both DNA-damaging agents and replication arrest. Interestingly, hsk1(ts) mutants released to the restrictive temperature after early S-phase arrest in hydroxyurea (HU) are able to complete bulk DNA synthesis but they nevertheless undergo an abnormal mitosis. These findings indicate a second role for hsk1 subsequent to HU arrest. Consistent with a later S-phase role, hsk1(ts) is synthetically lethal with Deltarqh1 (RecQ helicase) or rad21ts (cohesin) mutants and suppressed by Deltacds1 (RAD53 kinase) mutants. We demonstrate that Hsk1p undergoes Cds1p-dependent phosphorylation in response to HU and that it is a direct substrate of purified Cds1p kinase in vitro. These results indicate that the Hsk1p kinase is a potential target of Cds1p regulation and that its activity is required after replication initiation for normal mitosis.     

Nuclear localization of Schizosaccharomyces pombe Mcm2/Cdc19p requires MCM complex assembly

The minichromosome maintenance (MCM) proteins MCM2-MCM7 are conserved eukaryotic replication factors that assemble in a heterohexameric complex. In fission yeast, these proteins are nuclear throughout the cell cycle. In studying the mechanism that regulates assembly of the MCM complex, we analyzed the cis and trans elements required for nuclear localization of a single subunit, Mcm2p. Mutation of any single mcm gene leads to redistribution of wild-type MCM subunits to the cytoplasm, and this redistribution depends on an active nuclear export system. We identified the nuclear localization signal sequences of Mcm2p and showed that these are required for nuclear targeting of other MCM subunits. In turn, Mcm2p must associate with other MCM proteins for its proper localization; nuclear localization of MCM proteins thus requires assembly of MCM proteins in a complex. We suggest that coupling complex assembly to nuclear targeting and retention ensures that only intact heterohexameric MCM complexes remain nuclear.     

A new genetic method for isolating functionally interacting genes: high plo1(+)-dependent mutants and their suppressors define genes in mitotic and septation pathways in fission yeast

We describe a general genetic method to identify genes encoding proteins that functionally interact with and/or are good candidates for downstream targets of a particular gene product. The screen identifies mutants whose growth depends on high levels of expression of that gene. We apply this to the plo1(+) gene that encodes a fission yeast homologue of the polo-like kinases. plo1(+) regulates both spindle formation and septation. We have isolated 17 high plo1(+)-dependent (pld) mutants that show defects in mitosis or septation. Three mutants show a mitotic arrest phenotype. Among the 14 pld mutants with septation defects, 12 mapped to known loci: cdc7, cdc15, cdc11 spg1, and sid2. One of the pld mutants, cdc7-PD1, was selected for suppressor analysis. As multicopy suppressors, we isolated four known genes involved in septation in fission yeast: spg1(+), sce3(+), cdc8(+), and rho1(+), and two previously uncharacterized genes, mpd1(+) and mpd2(+). mpd1(+) exhibits high homology to phosphatidylinositol 4-phosphate 5-kinase, while mpd2(+) resembles Saccharomyces cerevisiae SMY2; both proteins are involved in the regulation of actin-mediated processes. As chromosomal suppressors of cdc7-PD1, we isolated mutations of cdc16 that resulted in multiseptation without nuclear division. cdc16(+), dma1(+), byr3(+), byr4(+) and a truncated form of the cdc7 gene were isolated by complementation of one of these cdc16 mutations. These results demonstrate that screening for high dose-dependent mutants and their suppressors is an effective approach to identify functionally interacting genes.     

Different phenotypes in vivo are associated with ATPase motif mutations in Schizosaccharomyces pombe minichromosome maintenance proteins

The six conserved MCM proteins are essential for normal DNA replication. They share a central core of homology that contains sequences related to DNA-dependent and AAA(+) ATPases. It has been suggested that the MCMs form a replicative helicase because a hexameric subcomplex formed by MCM4, -6, and -7 proteins has in vitro DNA helicase activity. To test whether ATPase and helicase activities are required for MCM protein function in vivo, we mutated conserved residues in the Walker A and Walker B motifs of MCM4, -6, and -7 and determined that equivalent mutations in these three proteins have different in vivo effects in fission yeast. Some mutations reported to abolish the in vitro helicase activity of the mouse MCM4/6/7 subcomplex do not affect the in vivo function of fission yeast MCM complex. Mutations of consensus CDK sites in Mcm4p and Mcm7p also have no phenotypic consequences. Co-immunoprecipitation analyses and in situ chromatin-binding experiments were used to study the ability of the mutant Mcm4ps to associate with the other MCMs, localize to the nucleus, and bind to chromatin. We conclude that the role of ATP binding and hydrolysis is different for different MCM subunits.     

Mutational analysis of conserved sequence motifs in the budding yeast Cdc6 protein

The Cdc6 protein is required to load a complex of Mcm2-7 family members (the MCM complex) into prereplicative complexes at budding yeast origins of DNA replication. Cdc6p is a member of the AAA(+) superfamily of proteins, which includes the prokaryotic and eukaryotic clamp loading proteins. These proteins share a number of conserved regions of homology and a common three-dimensional architecture. Two of the conserved sequence motifs are the Walker A and B motifs that are involved in nucleotide metabolism and are essential for Cdc6p function in vivo. Here, we analyse mutants in the other conserved sequence motifs. Several of these mutants are temperature-sensitive for growth and are unable to recruit the MCM complex to chromatin at the restrictive temperature. In one such temperature-sensitive mutant, a highly conserved asparagine residue in the sensor I motif was changed to alanine. Overexpression of this mutant protein is lethal. This phenotype is very similar to the phenotype previously described for a mutation in the Walker B motif, suggesting a common role for sensor I and the Walker B motif in Cdc6 function.     

Isolation of an essential Schizosaccharomyces pombe gene, prp31(+), that links splicing and meiosis

We carried out a screen for mutants that arrest prior to premeiotic S phase. One of the strains we isolated contains a temperature-sensitive allele mutation in the fission yeast prp31⁺ gene. The prp31-E1 mutant is defective in vegetative cell growth and in meiotic progression. It is synthetically lethal with prp6 and displays a pre-mRNA splicing defect at the restrictive temperature. We cloned the wild-type gene by complementation of the temperature-sensitive mutant phenotype. Prp31p is closely related to human and budding yeast PRP31 homologs and is likely to function as a general splicing factor in both vegetative growth and sexual differentiation.     

CDC37 is required for p60v-src activity in yeast.

Mutations in genes encoding the molecular chaperones Hsp90 and Ydj1p suppress the toxicity of the protein tyrosine kinase p60v-src in yeast by reducing its levels or its kinase activity. We describe isolation and characterization of novel p60v-src-resistant, temperature-sensitive cdc37 mutants, cdc37-34 and cdc37-17, which produce less p60v-src than the parental wild-type strain at 23 degrees C. However, p60v-src levels are not low enough to account for the resistance of these strains. Asynchronously growing cdc37-34 and cdc37-17 mutants arrest in G1 and G2/M when shifted from permissive temperatures (23 degrees C) to the restrictive temperature (37 degrees C), but hydroxyurea-synchronized cdc37-34 and cdc37-17 mutants arrest in G2/M when released from the hydroxyurea block and shifted from 23 to 37 degrees C. The previously described temperature-sensitive cdc37-1 mutant is p60v-src-sensitive and produces wild-type amounts of p60v-src at permissive temperatures but becomes p60v-src-resistant at its restrictive temperature, 38 degrees C. In all three cdc37 mutants, inactivation of Cdc37p by incubation at 38 degrees C reduces p60v-src-dependent tyrosine phosphorylation of yeast proteins to low or undetectable levels. Also, p60v-src levels are enriched in urea-solubilized extracts and depleted in detergent-solubilized extracts of all three cdc37 mutants prepared from cells incubated at the restrictive temperature. These results suggest that Cdc37p is required for maintenance of p60v-src in a soluble, biologically active form.     

Essential role of MCM proteins in premeiotic DNA replication

A critical event in eukaryotic DNA replication involves association of minichromosome maintenance (MCM2-7) proteins with origins, to form prereplicative complexes (pre-RCs) that are competent for initiation. The ability of mutants defective in MCM2-7 function to complete meiosis had suggested that pre-RC components could be irrelevant to premeiotic S phase. We show here that MCM2-7 proteins bind to chromatin in fission yeast cells preparing for meiosis and during premeiotic S phase in a manner suggesting they in fact are required for DNA replication in the meiotic cycle. This is confirmed by analysis of a degron mcm4 mutant, which cannot carry out premeiotic DNA replication. Later in meiosis, Mcm4 chromatin association is blocked between meiotic nuclear divisions, presumably accounting for the absence of a second round of DNA replication. Together, these results emphasize similarity between replication mechanisms in mitotic and meiotic cell cycles.