| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2000: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1999: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1998: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
Mcm1Op is an essential factor for the initiation of DNA replication in Saccharomyces cerevisiae. Mcm10p interacts physically and genetically with Mcm2-7 protein family and a lesion in MCM10 results in the pausing of elongation forks at replication origins which failed to be activated, suggesting that in addition to mediating the activation of replication initiation at replication origins, Mcm10p is required for the disassembly of the pre-replicative complex, which allows smooth passage of replication forks. Mcm10p is structurally conserved among eukaryotes like other replicatgion proteins.
We found that an mcm10 mutation causes loss fo chrommosome integrity during S phase and prevents entry into mitosis despite completion of chromosomal DNA replication. Furthermore, Mcm10p interacts genetically with origin recognition complex (ORC) and various replication elongation factors, including a subunit of DNA polymerase e and d.We also screened mutants that are synthetically lethal with mcm10. We obtained not only mutations in intiation factors, CDC45, MCM2, and MCM7, but also DNA2 which encodes an essential DNA helicase required for processing of Okazaki fragment.
Mcm10p is an abundant protein (approximately 4 x 10^4 copies per haploid cell) that is almost exclusively localized in the chromatin and/or nuclear matrix fraction during all phases of the cell cycle. When visualized by chromosome-spreading method followed by immunostaining, Mcm10p formed punctated foci on chromatin throughout the cell cycle and these foci mostly overlapped with those of Orc1p, a component of ORC.Furthermore, we found that Mcm10p physically associated with ORC by immunoprecipitation.
These results suggest that Mcm10p, like Mcm2-7 proteins, is a critical component of the pre-replication chromatin and acts together with ORC during the initiation of chromosomal DNA replication ; in addition, Mcm10p plays an essentail role during elongation of DNA replication.
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