| Budget Amount *help |
¥14,600,000 (Direct Cost: ¥14,600,000)
Fiscal Year 2006: ¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 2005: ¥8,200,000 (Direct Cost: ¥8,200,000)
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| Research Abstract |
Initiation of chromosome DNA replication in eukaryotes is tightly regulated through assembly of replication factors at replication origins. We here investigated dependence of the assembly of the initiation complex on particular factors using temperature-sensitive fission yeast mutants. The psf3-1 mutant, a GINS component mutant, arrested with un-replicated DNA at the restrictive temperature and the DNA content gradually increased, suggesting a defect in DNA replication. The mutation impaired GINS complex formation as shown by pull-down experiments. Chromatin immunoprecipitation assays indicated that GINS integrity was required for origin loading of Psf2, Cut5 and Cdc45, but not Sld3. In contrast, loading of Psf2 onto origins depended on Sld3 and Cut5 but not on Cdc45. These results suggest that Sld3 functions furthest upstream in initiation complex assembly, followed by GINS and Cut5, then Cdc45. Consistent with this conclusion, Cdc7-Dbf4 kinase (DDK) but not cyclin-dependent kinase (C
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DK) was required for Sld3 loading, while recruitment of the other factors depended on both kinases. These results suggest that DDK and CDK regulate distinct steps in activation of replication origins in fission yeast.
DNA replication of eukaryotic chromosomes initiates at a number of discrete loci, called replication origins. Distribution and regulation of origins are important for complete duplication of the genome. Here, we determined locations of Orcl and Mcm6, components of pre-replicative complex (pre-RC), on whole genome of Schizosaccharomyces pombe using a high-resolution tiling-array. Pre-RC sites were identified in 460 intergenic regions, where Orcl and Mcm6 colocalized. By mapping of 5-bromo-2'-deoxyuridine (BrdU)-incorporated DNA in the presence of hydroxyurea (HU), 307 pre-RC sites were identified as early-firing origins. In contrast, 153 pre-RC sites without BrdU incorporation were considered to be late and/or inefficient origins. Inactivation of replication checkpoint by Cds1 deletion resulted in BrdU incorporation with HU specifically at the late origins. Early and late origins tend to distribute separately in large chromosome regions. Interestingly, pericentromeric heterochromatin and the silent mating type locus replicated in the presence of HU, while the inner centromere or subtelomeric heterochromatin did not. Notably, MCM did not bind to inner centromeres where ORC was located. Thus, replication is differentially regulated in chromosome domains. Less
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