Biomedical subjects

Stephen J Aves

Publications and source records attributed to Stephen J Aves.

4 recordsLinked to original sources

PubMed2003

Fission yeast Cdc23/Mcm10 functions after pre-replicative complex formation to promote Cdc45 chromatin binding.

Using a cytological assay to monitor the successive chromatin association of replication proteins leading to replication initiation, we have investigated the function of fission yeast Cdc23/Mcm10 in DNA replication. Inactivation of Cdc23 before replication initiation using tight degron mutations has no effect on Mcm2 chromatin association, and thus pre-replicative complex (pre-RC) formation, although Cdc45 chromatin binding is blocked. Inactivating Cdc23 during an S phase block after Cdc45 has bound causes a small reduction in Cdc45 chromatin binding, and replication does not terminate in the absence of Mcm10 function. These observations show that Cdc23/Mcm10 function is conserved between fission yeast and Xenopus, where in vitro analysis has indicated a similar requirement for Cdc45 binding, but apparently not compared with Saccharomyces cerevisiae, where Mcm10 is needed for Mcm2 chromatin binding. However, unlike the situation in Xenopus, where Mcm10 chromatin binding is dependent on Mcm2-7, we show that the fission yeast protein is bound to chromatin throughout the cell cycle in growing cells, and only displaced from chromatin during quiescence. On return to growth, Cdc23 chromatin binding is rapidly reestablished independently from pre-RC formation, suggesting that chromatin association of Cdc23 provides a link between proliferation and competence to execute DNA replication.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2003

Genes encoding two essential DNA replication activation proteins, Cdc6 and Mcm3, exhibit very different patterns of expression in the tobacco BY-2 cell cycle.

Very little is known about the expression patterns in plants of genes that encode proteins involved in the initiation of DNA replication. Partial cDNA sequences that encode Cdc6 and Mcm3 in tobacco have been isolated. The sequences were used as probes in northern blots which suggested that, in the cell cycle of synchronized tobacco BY-2 cells, expression of CDC6 is confined to late G(1) and S-phase whereas the expression of MCM3 is not confined to any particular cell cycle phase. These data were confirmed and extended by real-time PCR measurements of mRNA abundance through the cell cycle. CDC6 exhibits a very clear peak of expression in S-phase whereas MCM3, expressed at a much lower level than CDC6, is not cell-cycle-regulated. These patterns of cell cycle gene expression resemble those found in the fission yeast Schizosaccharomyces pombe rather than those in budding yeast or mammalian cells.

Amino Acid Sequence↗

PubMed2002

Fission yeast Cdc23 interactions with DNA replication initiation proteins.

Schizosaccharomyces pombe Cdc23 is an essential DNA replication protein, conserved in eukaryotes and functionally homologous with Saccharomyces cerevisiae Dna43 (Mcm10). We sought evidence for interactions between Cdc23 and the MCM2-7 complex, a component of both the pre-replicative complex and the replication fork. Cdc23 shows genetic interactions with four MCM subunits: cdc23-M36 and cdc23-1E2 alleles both show synthetic phenotypes with mcm2 (cdc19-P1) and mcm6 (mis5-268), and cdc23-M36 is synthetically lethal with mcm4 (cdc21-K46) and with mcm5 (nda4-108). The wild-type cdc23 gene on multicopy plasmids can partially suppress temperature-dependent defects in mcm5 (nda4-108). Two-hybrid analysis demonstrates interactions at the protein-protein level between Cdc23 and Mcm4, Mcm5 and Mcm6. Cdc23 also interacts with four subunits of the Schizosaccharomyces pombe origin recognition complex (ORC) in yeast two-hybrid assay: Orc1, Orc2, Orc5 and Orc6. We found no evidence for interaction between Cdc23 and the MCM recruitment factor Cdc18 (the homologue of Saccharomyces cerevisiae Cdc6). Unlike Cdc18, Cdc23 mRNA shows no significant fluctuation in level through the cell cycle. These data suggest that fission yeast Cdc23 is an MCM-associated factor which has a role in the initiation of DNA replication.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2002

The mei3 region of the Schizosaccharomyces pombe genome.

Expression of the mei3 gene is sufficient to induce meiosis in the fission yeast Schizosaccharomyces pombe. The mei3 gene is located 0.64 Mb from the telomere of the left arm of Sz. pombe chromosome II. We have sequenced and analysed 107 kb of DNA from the mei3 genomic region. The sequence includes 14 known genes (bag1-B, csh3, dps1, gpt1, mei3, mfm3, pac1, prp31, rpl38-1, rpn3, rti1, spa1, spm1 and ubc4) and 26 other open reading frames (ORFs) longer than 100 codons: a density of one protein-coding gene per 2.7 kb. Twenty-one of the 40 ORFs (53%) have introns. In addition there is one lone Tf1 transposon long terminal repeat (LTR), tRNA(Trp) and tRNA(Ser) genes and a 5S rRNA gene. 14 of the novel ORFs show sequence similarities which suggest functions of their products, including a coatomer alpha-subunit, a catechol O-methyltransferase, protein kinase, asparagine synthetase, zinc metalloprotease, acetyltransferase, phosphatidylinositol 4-kinase, inositol polyphosphate phosphatase, GTPase-activating protein, permease, pre-mRNA splicing factor, 20S proteasome component and a thioredoxin-like protein. One predicted protein has similarity to the human Cockayne syndrome protein CSA and one with human GTPase XPA binding protein XAB1. Three ORFs are likely to code for proteins because they have sequence similarity with hypothetical proteins, three encode predicted coiled-coil proteins and four are sequence orphans.

Cosmids↗