Molecular cloning of cDNA encoding mouse Cdc21 and CDC46 homologs and characterization of the products: physical interaction between P1(MCM3) and CDC46 proteins.

Two new mouse genes encoding proteins that belong to the yeast minichromosome maintenance (MCM) protein family, which is involved in the initiation of DNA replication, were isolated and their nucleotide sequence was determined. They were a putative CDC46/MCM5 homolog and a putative cdc21 homolog. About 30% amino acid identity was obtained between members in the family, and > 40% between the putative mouse and yeast homologs. The expression of these genes was cell-cycle specific at the late G1 to S phase. Immunochemical analyses showed the physical interaction between mouse P1MCM3 and CDC46 protein. These results suggest that MCM proteins function in co-ordination for DNA replication.     

Identification of Cdc45p,an essential factor required for DNA replication

CDC45 is an essential gene required for initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. CDC45 interacts genetically with CDC46 and CDC47, both members of the MCM family of genes which have been implicated in the licensing of DNA replication. In this report, the isolation of CDC45 is described. The complementing gene is linked to an essential open reading frame on chromosome XII. CDC45 was found to be cell cycle regulated and steady-state mRNA levels are G1/S-specific. CDC45 encodes a protein structurally related to Tsd2p, a protein required for DNA replication in Ustilago maydis. CDC45 also interacts genetically with ORC2, the gene encoding the second subunit of the origin recognition complex, ORC, and MCM3, another member of the MCM family. The cdc45-1 mutant has a plasmid maintenance defect which is rescued by the addition of multiple potential origins to the plasmid.     

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.     

Decreased UV mutagenesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae.

Summary A DNA replication mutant of yeast, cdc8, was found to decrease UV-induced reversion of lys2-1, arg4-17, tyr1 and ura1. This effect was observed with all three alleles of cdc8 tested. Survival curves obtained following UV irradiation in cdc8 rad double mutants show that cdc8 is epistatic to rad6, as well as to rad1; cdc8 rad51 double mutants seem to be more sensitive than the single mutants. Since UV-induced reversion in cdc8 rad1 and cdc8 rad51 double mutants is like that of the cdc8 single mutants, we conclude that CDC8 plays a direct role in error-prone repair. To test whether CDC8 codes for a DNA polymerase, we have purified both DNA polymerase I and DNA polymerase II from cdc8 and CDC+ cells. The purified DNA polymerases from cdc8 were no more heat labile than those from CDC+, suggesting that CDC8 is not a structural gene for either enzyme.     

Genetic analysis of human p34CDC2 function in fission yeast

The p34^cdc2 protein kinase plays a key role in the control of the mitotic cell cycle of fission yeast, being required for both entry into S-phase and for entry into mitosis in the mitotic cell cycle, as well as for the initiation of the second meiotic nuclear division. In recent years, structural and functional homologues of p34^cdc2, as well as several of the proteins that interact with and regulate p34^cdc2 function in fission yeast, have been identified in a wide range of higher eukaryotic cell types, suggesting that the control mechanisms uncovered in this simple eukaryote are likely to be well conserved across evolution. Here we describe the construction and characterisation of a fission yeast strain in which the endogenous p34^cdc2 protein is entirely absent and is replaced by its human functional homologue p34^CDC2, We have used this strain to analyse aspects of the function of the human p34^CDC2 protein genetically. We show that the function of the human p34^CDC2 protein in fission yeast cells is dependent upon the action of the protein tyrosine phosphatase p80^cdc25 that it responds to altered levels of both the mitotic inhibitor p107²³³¹ and the p34^cdc2-binding protein p13^suc1, and is lethal in combination with the mutant B-type cyclin p56^cdc13-117. In addition, we demonstrate that the human p34^CDC2 protein is proficient for fission yeast meiosis, and examine the behaviour of two mutant p34^CDC2 proteins in fission yeast.     

Genetic analysis of human p34CDC2 function in fission yeast

The p34^cdc2 protein kinase plays a key role in the control of the mitotic cell cycle of fission yeast, being required for both entry into S-phase and for entry into mitosis in the mitotic cell cycle, as well as for the initiation of the second meiotic nuclear division. In recent years, structural and functional homologues of p34^cdc2, as well as several of the proteins that interact with and regulate p34^cdc2 function in fission yeast, have been identified in a wide range of higher eukaryotic cell types, suggesting that the control mechanisms uncovered in this simple eukaryote are likely to be well conserved across evolution. Here we describe the construction and characterisation of a fission yeast strain in which the endogenous p34^cdc2 protein is entirely absent and is replaced by its human functional homologue p34^CDC2, We have used this strain to analyse aspects of the function of the human p34^CDC2 protein genetically. We show that the function of the human p34^CDC2 protein in fission yeast cells is dependent upon the action of the protein tyrosine phosphatase p80^cdc25 that it responds to altered levels of both the mitotic inhibitor p107²³³¹ and the p34^cdc2-binding protein p13^suc1, and is lethal in combination with the mutant B-type cyclin p56^cdc13-117. In addition, we demonstrate that the human p34^CDC2 protein is proficient for fission yeast meiosis, and examine the behaviour of two mutant p34^CDC2 proteins in fission yeast.     

Fission yeast cdc21+ belongs to a family of proteins involved in an early step of chromosome replication.

The cdc21+ gene of Schizosaccharomyces pombe was originally identified in a screen for cdc mutants affecting S phase and nuclear division. Here we show that the cdc21+ gene product belongs to a family of proteins implicated in DNA replication. These include the Saccharomyces cerevisiae MCM2 and MCM3 proteins, which are needed for the efficient function of certain replication origins, and S.cerevisiae CDC46, which is required for the initiation of chromosome replication. The cdc21 mutant is defective in the mitotic maintenance of some plasmids, like mcm2 and mcm3. The mutant arrests with a single nucleus containing two genome equivalents of DNA, and maintains a cytoplasmic microtubular configuration. Activation of most, but not all, replication origins in the mutant may result in failure to replicate a small proportion of the genome, and this could explain the arrest phenotypes. Using the polymerase chain reaction technique, we have identified new cdc21(+)-related genes in S.cerevisiae, S.pombe and Xenopus laevis. Our results suggest that individual members of the cdc21(+)-related family are highly conserved in evolution.     

The structure and function of MCM from archaeal M. Thermoautotrophicum

Eukaryotic chromosomal DNA is licensed for replication precisely once in each cell cycle. The mini-chromosome maintenance (MCM) complex plays a role in this replication licensing. We have determined the structure of a fragment of MCM from Methanobacterium thermoautotrophicum (mtMCM), a model system for eukaryotic MCM. The structure reveals a novel dodecameric architecture with a remarkably long central channel. The channel surface has an unusually high positive charge and binds DNA. We also show that the structure of the N-terminal fragment is conserved for all MCMs proteins despite highly divergent sequences, suggesting a common architecture for a similar task: gripping/remodeling DNA and regulating MCM activity. An mtMCM mutant protein equivalent to a yeast MCM5 (CDC46) protein with the bob1 mutation at its N terminus has only subtle structural changes, suggesting a Cdc7-bypass mechanism by Bob1 in yeast. Yeast bypass experiments using MCM5 mutant proteins support the hypothesis for the bypass mechanism.     

The role ofSaccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance

Successful progression through the cell cycle requires the coupling of mitotic spindle formation to DNA replication. In this report we present evidence suggesting that, inSaccharomyces cerevisiae, theCDC40 gene product is required to regulate both DNA replication and mitotic spindle formation. The deduced amino acid sequence ofCDC40 (455 amino acids) contains four copies of a -transducin-like repeat. Cdc40p is essential only at elevated temperatures, as a complete deletion or a truncated protein (deletion of the C-terminal 217 amino acids in thecdc40-1 allele) results in normal vegetative growth at 23°C, and cell cycle arrest at 36°C. In the mitotic cell cycle Cdc40p is apparently required for at least two steps: (1) for entry into S phase (neither DNA synthesis, nor mitotic spindle formation occurs at 36°C and (2) for completion of S-phase (cdc40::LEU2 cells cannot complete the cell cycle when returned to the permissive temperature in the presence of hydroxyurea). The role of Cdc40p as a regulatory protein linking DNA synthesis, spindle assembly/maintenance, and maturation promoting factor (MPF) activity is discussed.     

Fission yeast cdc21, a member of the MCM protein family, is required for onset of S phase and is located in the nucleus throughout the cell cycle.

The fission yeast cdc21 protein belongs to the MCM family, implicated in the once per cell cycle regulation of chromosome replication. In budding yeast, proteins in this family are eliminated from the nucleus during S phase, which has led to the suggestion that they may serve to distinguish unreplicated from replicated DNA, as in the licensing factor model. We show here that, in contrast to the situation in budding yeast, cdc21 remains in the nucleus after S phase, as is found for related proteins in mammalian cells. We suggest that regulation of nuclear import of these proteins may not be an essential aspect of their function in chromosome replication. To determine the function of cdc21+, we have analysed the phenotype of a gene deletion. cdc21+ is required for entry into S phase and, unexpectedly, a proportion of cells depleted of the gene product are able to enter mitosis in the absence of DNA replication. These results are consistent with the view that individual proteins in the MCM family are required for all initiation events, and defective initiation may impair the coordination between mitosis and S phase.     

Evolutionary conservation of the human homologue of the yeast cell cycle control gene cdc2 and assignment of Cd2 to chromosome 10

Summary The human homologue of the fission yeast Schizosaccharomyces pombe cell cycle control gene cdc2 has been assigned to chromosome 10. DNA hybridization reveals that this gene is highly conserved in vertebrates. The human CDC2 gene probe detects a simple two-allele polymorphism in Taq1-digested DNA.     

The identification of a Caenorhabditis elegans homolog of p34cdc2 kinase

We have identified a Caenorhabditis elegans homolog of p34^cdc2 kinase. The C. elegans homolog, ncc-1, is -60% identical to p34^cdc2 of Homo sapiens. When expressed from a constitutive yeast promoter, ncc-1 is capable of complementing a conditional lethal mutation in the CDC28 gene of Saccharomyces cerevisiae, indicating that this C. elegans homolog can properly regulate the cell cycle.     

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     

Identification and functional characterization of <Emphasis Type="Italic">Candida albicans CDC4</Emphasis>

Summary The CDC4 gene of Saccharomyces cerevisiae encodes an essential function that is required for G1-S and G2-M transitions during mitosis and at various stages during meiosis. We have isolated a functional homologue of CDC4 (CaCDC4) from the pathogenic yeast Candida albicans by complementing the S. cerevisiae cdc4-3 mutation with CaCDC4 expressed from its own promoter on a single-copy vector. The predicted product of CaCDC4 has 37% overall identity to the S. cerevisiae Cdc4 protein, although this identity is biased towards the C-terminal region of the two proteins which contains eight copies of the degenerate WD-40 motif, an element found in proteins that regulate diverse biological processes and an F-box domain proximal to the first iteration of the WD-40 motif. Both the F-box domain and WD-40 motifs appear necessary for the mitotic functions of Cdc4 in both yeasts. In contrast to its conserved role in mitosis, C. albicans CDC4 is unable to rescue the meiotic deficiency in a S. cerevisiae cdc4 homozygous diploid under restrictive conditions, even when expressed from an efficient S. cerevisiae promoter. In opposition to S. cerevisiae CDC4 being essential, C. albicans CDC4 appears to be nonessential and in its absence is critical for filamentous growth in C. albicans.     

An improved assay for DNA ligase reveals temperature-sensitive activity in cdc9 mutants of Saccharomyces cerevisiae

Summary A sensitive and quantitative assay for DNA ligase has been developed which is suitable for the analysis of crude cell extracts of yeast. The assay is sufficiently sensitive to detect the low levels of DNA ligase activity remaining in cdc9 mutants of Saccharomyces cerevisiae. Indeed, we have been able to show that this residual activity is temperature-sensitive, thus establishing finally that CDC9 is the structural gene for DNA ligase.     

High abundance of CDC45 inhibits cell proliferation through elevation of HSPA6

ObjectivesCDC45 is the core component of CMG (CDC45‐MCMs‐GINS) complex that plays important role in the initial step of DNA replication in eukaryotic cells. The expression level of cdc45 is under the critical control for the accurate cell cycle progression. Loss‐of‐function of cdc45 has been demonstrated to inhibit cell proliferation and leads to cell death due to the inhibition of DNA replication and G1‐phase arrest. An increasing of CDC45 inhibits cell proliferation as well. Nevertheless, a systematic analysis of the effect of high dose of CDC45 on cell physiology and behaviors is unclear. In the present study, we aimed to investigate the effects and mechanisms of high dose of CDC45 on cell behaviors.Materials and MethodsWe overexpressed cdc45 in cultured cell lines, Ciona and Drosophila embryos, respectively. The cell cycle progression was examined by the BrdU incorporation experiment, flow cytometry and PH3 (phospho‐Histone 3) staining. RNA‐sequencing analysis and qRT‐PCR were carried out to screen the affected genes in HeLa cells overexpressing cdc45. siRNA‐mediated knockdown was performed to investigate gene functions in HeLa cells overexpressing cdc45.ResultsWe found that high level of cdc45 from different species (human, mammal, ascidian, and Drosophila) inhibited cell cycle in vitro and in vivo. High dose of CDC45 blocks cells entering into S phase. However, we failed to detect DNA damage and cell apoptosis. We identified hspa6 was the most upregulated gene in HeLa cells overexpressing cdc45 via RNA‐seq analysis and qRT‐PCR validation. Overexpression of Hs‐hspa6 inhibited proliferation rate and DNA replication in HeLa cells, mimicking the phenotype of cdc45 overexpression. RNAi against hspa6 partially rescued the cell proliferation defect caused by high dose of CDC45.ConclusionsOur study suggests that high abundance of CDC45 stops cell cycle. Instead of inducing apoptosis, excessive CDC45 prevents cell entering S phase probably due to promoting hspa6 expression.

CDC45 is essential for DNA replication. Surprisingly high dose of CDC45 inhibits cell proliferation and blocks cell entering S phase without inducing apoptosis nor aneuploidy as expected. The overexpressed CDC45 induces the elevation of HSPA6, which in turn inhibits cell proliferation.     

Mutations in CDC14 result in high sensitivity to cyclin gene dosage in Saccharomyces cerevisiae

We screened for mutations that resulted in lethality when the G1 cyclin Cln2p was overexpressed throughout the cell cycle in Saccharomyces cerevisiae. Mutations in five complementation groups were found to give this phenotype, and three of the mutated genes were identified as MEC1, NUP170, and CDC14. Mutations in CDC14 may have been recovered in the screen because Cdc14p may reduce the cyclin B (Clb)-associated Cdc28 kinase activity in late mitosis, and Cln2p may normally activate Clb-Cdc28 kinase activity by related mechanisms. In agreement with the idea that cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality with cdc14-1, as did the deletion of HCT1, which is required for proteolysis of Clb2p. Surprisingly, deletion of the gene for the major B-type cyclin, CLB2, also caused synthetic lethality with the cdc14-1 mutation. The clb2 cdc14 strains arrested with replicated but unseparated DNA and unseparated spindle pole bodies; this phenotype is distinct from the late mitotic arrest of the sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14 CLN2 overexpressor. We found genetic interactions between CDC14 and the replication initiator gene CDC6, extending previous observations of interactions between the late mitotic function of Cdc14p and control of DNA replication. We also describe genetic interactions between CDC28 and CDC14. Received: 24 May 1999 / Accepted: 19 October 1999