Overexpression of a novel gene, Cms1, can rescue the growth arrest of a Saccharomyces cerevisiae mcm10 suppressor.

MCM10 protein is an essential replication factor involved in the initiation of DNA replication. A mcm10 mutant (mcm10-1) of budding yeast shows a growth arrest at 37 degrees C. In the present work, we have isolated a mcm10-1 suppressor strain, which grows at 37 degrees C. Interestingly, this mcm10-1 suppressor undergoes cell cycle arrest at 14 degrees C. A novel gene, YLR003c, is identified by high-copy complementation of this suppressor. We called it as Cms1 (Complementation of Mcm 10 Suppressor). Furthermore, the experiments of transformation show that cells of mcm10-1 suppressor with high-copy plasmid but not low-copy plasmid grow at 14 degrees C, indicating that overexpression of Cms1 can rescue the growth arrest of this mcm10 suppressor at non-permissive temperature. These results suggest that CMS1 protein may functionally interact with MCM10 protein and play a role in the regulation of DNA replication and cell cycle control.     

The phenotype of the minichromosome maintenance mutant mcm3 is characteristic of mutants defective in DNA replication.

MCM3 is an essential gene involved in the maintenance of minichromosomes in yeast cells. It encodes a protein of 971 amino acids that shows striking homology to the Mcm2 protein. We have mapped the mcm3-1 mutation of the left arm of chromosome V approximately 3 kb centromere proximal of anp1. The mcm3-1 mutant was found to be thermosensitive for growth. Under permissive growth conditions, it was defective in minichromosome maintenance in an autonomously replicating sequence-specific manner and showed an increase in chromosome loss and recombination. Under nonpermissive conditions, mcm3-1 exhibited a cell cycle arrest phenotype, arresting at the large-bud stage with an undivided nucleus that had a DNA content of nearly 2n. These phenotypes are consistent with incomplete replication of the genome of the mcm3-1 mutant, possibly as a result of limited replication initiation at selective autonomously replicating sequences leading to cell cycle arrest before mitosis. The phenotype exhibited by the mcm3 mutant is very similar to that of mcm2, suggesting that the Mcm2 and Mcm3 protein may play interacting roles in DNA replication.     

Characterization of Schizosaccharomyces pombe mcm7(+) and cdc23(+) (MCM10) and interactions with replication checkpoints

MCM proteins are required for the proper regulation of DNA replication. We cloned fission yeast mcm7(+) and showed it is essential for viability; spores lacking mcm7(+) begin S phase later than wild-type cells and arrest with an apparent 2C DNA content. We isolated a novel temperature-sensitive allele, mcm7-98, and also characterized two temperature-sensitive alleles of the fission yeast homolog of MCM10, cdc23(+). mcm7-98 and both cdc23ts alleles arrest with damaged chromosomes and an S phase delay. We find that mcm7-98 is synthetically lethal with the other mcmts mutants but does not interact genetically with either cdc23ts allele. However, cdc23-M36 interacts with mcm4ts. Unlike other mcm mutants or cdc23, mcm7-98 is synthetically lethal with checkpoint mutants Deltacds1, Deltachk1, or Deltarad3, suggesting chromosomal defects even at permissive temperature. Mcm7p is a nuclear protein throughout the cell cycle, and its localization is dependent on the other MCM proteins. Our data suggest that the Mcm3p-Mcm5p dimer interacts with the Mcm4p-Mcm6p-Mcm7p core complex through Mcm7p.     

Mcm3 is polyubiquitinated during mitosis before establishment of the pre-replication complex

To ensure fidelity in genome duplication, eukaryotes restrict DNA synthesis to once every cell division by a cascade of regulated steps. Central to this cascade is the periodic assembly of the hexameric MCM2-7 complex at replication origins. However, in Saccharomyces cerevisiae, only a fraction of each MCM protein is able to assemble into hexamers and associate with replication origins during M phase, suggesting that MCM complex assembly and recruitment may be regulated post-translationally. Here we show that a small fraction of Mcm3p is polyubiquitinated at the onset of MCM complex assembly. Reducing the rate of ubiquitination by uba1-165, a suppressor of mcm3-10, restored the interaction of Mcm3-10p with subunits of the MCM complex and its recruitment to the replication origin. Possible roles for ubiquitinated Mcm3p in the assembly of the MCM complex at replication origins are discussed.     

A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks through chromosomal replication origins in Saccharomyces cerevisiae.

We describe a new minichromosome maintenance factor, Mcm10, and show that this essential protein is involved in the initiation of DNA replication in Saccharomyces cerevisiae. The mcm10 mutant has an autonomously replicating sequence-specific minichromosome maintenance defect and arrests at the nonpermissive temperature with dumbbell morphology and 2C DNA content. Mcm10 is a nuclear protein that physically interacts with several members of the MCM2-7 family of DNA replication initiation factors. Cloning and sequencing of the MCM10 gene show that it is identical to DNA43, a gene identified independently for its putative role in replicating DNA. Two-dimensional DNA gel analysis reveals that the mcm10-1 lesion causes a dramatic reduction in DNA replication initiation at chromosomal origins, including ORI1 and ORI121. Interestingly, the mcm10-1 lesion also causes replication forks to pause during elongation through these same loci. This novel phenotype suggests a unique role for the Mcm10 protein in the initiation of DNA synthesis at replication origins.     

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.     

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.     

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.     

Two mcm3 mutations affect different steps in the initiation of DNA replication

Mcm3 is a subunit of the hexameric MCM2-7 complex required for the initiation and elongation of DNA replication in eukaryotes. We have characterized two mutant alleles, mcm3-1 and mcm3-10, in Saccharomyces cerevisiae and showed that they are defective at different steps of the replication initiation process. Mcm3-10 contains a P118L substitution that compromises its interaction with Mcm5 and the recruitment of Mcm3 and Mcm7 to a replication origin. P118 is conserved between Mcm3, Mcm4, Mcm5, and Mcm7. An identical substitution of this conserved residue in Mcm5 (P83L of mcm5-bob1) strengthens the interaction between Mcm3 and Mcm5 and allows cells to enter S phase independent of Cdc7-Dbf4 kinase (Hardy, C. F., Dryga, O., Pahl, P. M. B., and Sclafani, R. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3151-3155). Mcm3-1 contains a G246E mutation that diminishes the efficiency of replication initiation (Yan, H., Merchant, A. M., and Tye, B. K. (1993) Genes Dev. 7, 2149-2160) but not its interaction with Mcm5 or recruitment of the MCM2-7 complex to replication origin. These observations indicate that Mcm3-10 is defective in a step before, and Mcm3-1 is defective in a step after the recruitment of the MCM2-7 complex to replication origins.     

Dynamics of DNA binding of replication initiation proteins during de novo formation of pre-replicative complexes in Xenopus egg extracts

We investigated the dynamics of DNA binding of replication initiation proteins during formation of the pre-replicative complex (pre-RC) on plasmids in Xenopus egg extracts. The pre-RC was efficiently formed on plasmids at 23 degrees C, with one or a few origin recognition complex (ORC) molecules and approximately 10-20 mini-chromosome maintenance 2 (MCM2) molecules loaded onto each plasmid. Although geminin inhibited MCM loading, MCM interacted weakly but stoichiometrically with the plasmid in an ORC-dependent manner, even in the presence of geminin (with approximately 10 MCM2 molecules per plasmid). Interestingly, DNA binding of ORC, CDC6, and CDT1 was significantly stabilized in the presence of geminin, under which conditions approximately 10-20 molecules each of ORC and CDC6 were bound. Moreover, a similarly stable ORC-CDC6-CDT1 complex rapidly formed on DNA at lower temperature (0 degrees C) without geminin, with approximately 10-20 molecules each of ORC and CDC6 bound to the plasmid, but almost no binding of MCM. However, upon shifting the temperature to 23 degrees C, most ORC, CDC6, and CDT1 molecules were displaced from the DNA, leaving about one ORC molecule on the plasmid, whereas approximately 10 MCM2 molecules were loaded onto each plasmid. Furthermore, it was possible to load MCM onto DNA when the isolated ORC-CDC6-CDT1-DNA complex was mixed with purified MCM proteins. These results suggest that an ORC-CDC6-CDT1 complex pre-formed on DNA is directly involved in MCM loading and imply that each DNA-bound ORC molecule loads only one or a few MCM2-7 complexes during metazoan pre-RC formation.     

Replication of chromosomal DNA in Chinese hamster cells, cultivated at different temperatures

DNA fiber autoradiography was used to measure the rate of replication and the size of replication units in Chinese hamster cells cultured at 34, 37 and 39 degrees C. The average rate of DNA replication per single fork is 0.6 mcm/min at 34 degrees and 0.8 mcm/min at 37 and 39 degrees. In contrast to the change in the rate of DNA replication, no change was found in the size of replication units, which are more than 200 mcm at 31, 37 and 39 degrees. The change of the length of the S phase at various temperatures is determined only by the rate of DNA replication.     

Phosphorylation of MCM4 at sites inactivating DNA helicase activity of the MCM4-MCM6-MCM7 complex during Epstein-Barr virus productive replication

Induction of Epstein-Barr virus (EBV) lytic replication blocks chromosomal DNA replication notwithstanding an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. We report here that the phosphorylated form of MCM4, a subunit of the MCM complex essential for chromosomal DNA replication, increases with progression of lytic replication, Thr-19 and Thr-110 being CDK2/CDK1 targets whose phosphorylation inactivates MCM4-MCM6-MCM7 (MCM4-6-7) complex-associated DNA helicase. Expression of EBV-encoded protein kinase (EBV-PK) in HeLa cells caused phosphorylation of these sites on MCM4, leading to cell growth arrest. In vitro, the sites of MCM4 of the MCM4-6-7 hexamer were confirmed to be phosphorylated with EBV-PK, with the same loss of helicase activity as with CDK2/cyclin A. Introducing mutations in the N-terminal six Ser and Thr residues of MCM4 reduced the inhibition by CDK2/cyclin A, while EBV-PK inhibited the helicase activities of both wild-type and mutant MCM4-6-7 hexamers, probably since EBV-PK can phosphorylate MCM6 and another site(s) of MCM4 in addition to the N-terminal residues. Therefore, phosphorylation of the MCM complex by redundant actions of CDK and EBV-PK during lytic replication might provide one mechanism to block chromosomal DNA replication in the infected cells through inactivation of DNA unwinding by the MCM4-6-7 complex.     

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.     

Differential requirement of DNA replication factors for subtelomeric ARS consensus sequence protosilencers in Saccharomyces cerevisiae

The establishment of silent chromatin requires passage through S-phase, but not DNA replication per se. Nevertheless, many proteins that affect silencing are bona fide DNA replication factors. It is not clear if mutations in these replication factors affect silencing directly or indirectly via deregulation of S-phase or DNA replication. Consequently, the relationship between DNA replication and silencing remains an issue of debate. Here we analyze the effect of mutations in DNA replication factors (mcm5-461, mcm5-1, orc2-1, orc5-1, cdc45-1, cdc6-1, and cdc7-1) on the silencing of a group of reporter constructs, which contain different combinations of "natural" subtelomeric elements. We show that the mcm5-461, mcm5-1, and orc2-1 mutations affect silencing through subtelomeric ARS consensus sequences (ACS), while cdc6-1 affects silencing independently of ACS. orc5-1, cdc45-1, and cdc7-1 affect silencing through ACS, but also show ACS-independent effects. We also demonstrate that isolated nontelomeric ACS do not recapitulate the same effects when inserted in the telomere. We propose a model that defines the modes of action of MCM5 and CDC6 in silencing.