No abstract

No abstract

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.DNA replication is a fundamental process of all dividing cells. During the eukaryotic cell cycle, DNA replication occurs exactly once and is initiated only upon the completion of mitosis. Strict regulation of timing appears to be mediated through the coordinated activities of numerous proteins. Due in large part to studies of viral DNA replication, the enzymatic activities at the replication fork were elucidated many years ago (33). In contrast, trans-acting factors which regulate the initiation of DNA replication have been described only recently. In an effort to gain a comprehensive understanding of the factors involved in this essential process, we have sought to identify gene products that influence the initiation of DNA replication.Saccharomyces cerevisiae provides an excellent eukaryotic model for identifying proteins involved in DNA replication. Many replication initiation factors currently under investigation are conserved in mammalian cells and were found initially in yeast. Members of the origin recognition complex (ORC) (20,22) were originally identified biochemically through their binding to the consensus sequence of autonomously replicating sequences (ARSs), which function as DNA replication origins (2). Our screen for yeast minichromosome maintenance (mcm) mutants (39) has also been fruitful in identifying replication initiation factors, such as those of the MCM2-7 family (9, 29, 52). The MCM2-7 proteins are a family of six conserved proteins that are ubiquitous in eukaryotes. Their essential role in the initiation of DNA synthesis not only has been demonstrated by in vivo studies in a number of organisms, including S. cerevisiae (55), Schizosaccharomyces pombe (40), and Drosophila melanogaster (51), but also is supported by in vitro studies in Xenopus laevis (8,34,38). The MCM2-7 proteins interact with one another, and possibly other proteins, to form large complexes (35). Despite their structural similarity, each of these proteins is indispensable for ...

Meiosis ensures genetic diversification of gametes and sexual reproduction. For successful meiosis, multiple events such as DNA replication, recombination, and chromosome segregation must occur coordinately in a strict regulated order. We investigated the meiotic roles of Cdc7 kinase in the initiation of meiotic recombination, namely, DNA double-strand breaks (DSBs) mediated by Spo11 and other coactivating proteins. Genetic analysis using bob1-1 cdc7⌬ reveals that Cdc7 is essential for meiotic DSBs and meiosis I progression. We also demonstrate that the N-terminal region of Mer2, a Spo11 ancillary protein required for DSB formation and phosphorylated by cyclin-dependent kinase (CDK), contains two types of Cdc7-dependent phosphorylation sites near the CDK site (Ser30): One (Ser29) is essential for meiotic DSB formation, and the others exhibit a cumulative effect to facilitate DSB formation. Importantly, mutations on these sites confer severe defects in DSB formation even when the CDK phosphorylation is present at Ser30. Diploids of cdc7⌬ display defects in the chromatin binding of not only Spo11 but also Rec114 and Mei4, other meiotic coactivators that may assist Spo11 binding to DSB hot spots. We thus propose that Cdc7, in concert with CDK, regulates Spo11 loading to DSB sites via Mer2 phosphorylation.[Keywords: Cdc7; Mer2; meiotic recombination; Spo11; pre-DSB complex] Supplemental material is available at http://www.genesdev.org.

Saccharomyces cerevisiae POL2 encodes the catalytic subunit of DNA polymerase ⑀. This study investigates the cellular functions performed by the polymerase domain of Pol2p and its role in DNA metabolism. The pol2-16 mutation has a deletion in the catalytic domain of DNA polymerase ⑀ that eliminates its polymerase and exonuclease activities. It is a viable mutant, which displays temperature sensitivity for growth and a defect in elongation step of chromosomal DNA replication even at permissive temperatures. This mutation is synthetic lethal in combination with temperature-sensitive mutants or the 3-to 5-exonuclease-deficient mutant of DNA polymerase ␦ in a haploid cell. These results suggest that the catalytic activity of DNA polymerase ⑀ participates in the same pathway as DNA polymerase ␦, and this is consistent with the observation that DNA polymerases ␦ and ⑀ colocalize in some punctate foci on yeast chromatids during S phase. The pol2-16 mutant senesces more rapidly than wild type strain and also has shorter telomeres. These results indicate that the DNA polymerase domain of Pol2p is required for rapid, efficient, and highly accurate chromosomal DNA replication in yeast.Saccharomyces cerevisiae has three DNA polymerases (pol␣, -␦, and -⑀) 1 that are required for cell growth, chromosomal DNA replication (1), and DNA double-strand break repair (2). pol␣ consists of four subunits (Pol1p (Cdc17p), Pol10p, Pri1p, and Pri2p) and is primarily involved in the initiation of DNA replication and priming of Okazaki fragments. pol␦ and -⑀ are required during synthesis of the leading and lagging strands at the replication fork, binding at/or near replication origins, and moving along DNA with the replication fork (3, 4). The precise roles of pol␦ and pol⑀ during leading and lagging strand synthesis have yet not been defined; however, genetic and biochemical evidence suggests that lagging strand synthesis is carried out by pol␣ and pol␦ (5, 6). Nevertheless, simian virus 40 DNA replication only requires pol␣ and pol␦ (5).S. cerevisiae pol␦ contains the three subunits Pol3 (Cdc2), Hys2 (Pol31) (7), and Pol32 (8), which are homologues of Schizosaccharomyces pombe Pol3, Cdc1, and Cdc27, respectively. S. pombe pol␦ contains one additional subunit, Cmt1 (9). Purified yeast pol␦ requires accessory factors including PCNA and the RF-C to catalyze processive DNA synthesis; this suggests that pol␦ may be the leading strand DNA polymerase (5, 10). pol␦ has a 3Ј-to 5Ј-exonuclease, which acts as a proofreading/editing polymerase during DNA synthesis (11, 12).S. cerevisiae pol⑀ is also a multisubunit complex consisting of Pol2p, Dpb2p, Dpb3p, and Dpb4p (13,14). pol⑀ requires PCNA and RF-C complex to catalyze processive DNA synthesis on singly primed single-stranded viral DNA, although pol⑀ is a highly processive enzyme (13,15,16). Pol2p is the catalytic subunit of pol⑀, and it is encoded by the POL2 gene (17), which is essential in yeast. pol⑀ is a class B polymerase, characterized by six conserved domains (I-VI) in the N-terminal ha...

Background: MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Previous work showed that Mcm10p interacts with the Mcm2±7 protein complex that may be functioning as the replication-licensing factor. In addition, Mcm10p is required during origin activation and disassembly of the prereplicative complex, which allows smooth passage of replication forks.

Replication of mini F plasmid requires the plasmid-encoded RepE initiator protein and several host factors including DnaJ, DnaK, and GrpE, heat shock proteins of Escherchia coli. The RepE protein plays a crucial role in replication and exhibits two major functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription. One of the mini-F plasmid mutants that can replicate in the dnaj-defective host produces an altered RepE (RepE54) with a markedly enhanced initiator activity but little or no repressor activity. RepE54 has been purified from cell extracts primarily in monomeric form, unlike the wild-type RepE that is recovered in dlmeric form. Gel-retardation assays revealed that RepE54 monomers bind to ori2 (direct repeats) with a very high efficiency but hardly bind to the repE operator (inverted repeat), in accordance with the properties of RepE54 in vivo. Furthermore, the treatment of wild-type RepE dimers with protein denaturants enhanced their binding to ori2 but reduced binding to the operator: RepE dimers were partially converted to monomers, and the ori2 binding activity was uniquely associated with monomers. These results strongly suggest that RepE monomers represent an active form by binding to ori2 to initiate replication, whereas dimers act as an autogenous repressor by binding to the operator. We propose that RepE is structurally and functionally differentiated and that monomerization ofRepE dimers, presumably mediated by heat shock protein(s), activates the initiator function and participates in regulation of mini-F DNA replication.The mini-F plasmid, derived from the F (fertility) factor, replicates as a low-copy plasmid (one to two copies per host chromosome) in Escherichia coli (1, 2). The plasmid-encoded RepE initiator protein plays an essential and a specific role in initiating replication from the origin, ori2 (3-6). RepE exhibits two major functions: initiation of DNA replication from ori2 (initiator function) and autogenous repression of repE transcription (repressor function) (7,8). These functions of RepE require its binding to the four 19-bp direct repeat sequences (iterons) found within ori2 and to the repE promoter/operator, which contains an inverted repeat sequence (9-13); the half-sequence (10 bp) of the latter is similar (8-bp matches) to the 19-bp repeats (14). RepE has been purified as a dimer in several laboratories (11-13) and its binding to ori2 iterons and the operator was demonstrated by using DNase I footprinting (9, 10) and gel-retardation (10-13) assays. Thus, RepE dimers have been thought to be involved in binding to both DNA regions, but the structural basis for each of the specific functions of RepE remained undetermined.Besides RepE, several host factors including those involved in chromosomal DNA replication are required for mini-F plasmid replication. In particular, the heat shock oa factor (v.32), which is essential for transcription of repE (8,15), and a subset of heat shock proteins (DnaJ, DnaK, and GrpE) actively...

DNA polymerase epsilon (Polepsilon) of Saccharomyces cerevisiae is purified as a complex of four polypeptides with molecular masses of >250, 80, 34 (and 31) and 29 kDa as determined by SDS-PAGE. The genes POL2, DPB2 and DPB3, encoding the catalytic Pol2p, the second (Dpb2p) and the third largest subunits (Dpb3p) of the complex, respectively, were previously cloned and characterised. This paper reports the partial amino acid sequence of the fourth subunit (Dpb4p) of Polepsilon. This protein sequence matches parts of the predicted amino acid sequence from the YDR121w open reading frame on S.cerevisiae chromosome IV. Thus, YDR121w was renamed DPB4. A deletion mutant of DPB4 (Deltadpb4) is not lethal, but chromosomal DNA replication is slightly disturbed in this mutant. A double mutant haploid strain carrying the Deltadpb4 deletion and either pol2-11 or dpb11-1 is lethal at all temperatures tested. Furthermore, the restrictive temperature of double mutants carrying Deltadpb4 and dpb2-1, rad53-1 or rad53-21 is lower than in the corresponding single mutants. These results strongly suggest that Dpb4p plays an important role in maintaining the complex structure of Polepsilon in S.cerevisiae, even if it is not essential for cell growth. Structural homologues of DPB4 are present in other eukaryotic genomes, suggesting that the complex structure of S. cerevisiae Polepsilon is conserved in eukaryotes.