Regulation of chromosome DNA replication for genome integrity.

真核生物染色体DNA复制的起始是通过复制起始点的复制因子组装而受到严格调控的。我们在此利用温度敏感的裂变酵母突变体研究了起始复合物的组装对特定因素的依赖性。psf3-1突变体是一个GINS组分突变体，在限制温度下被未复制的DNA捕获，DNA含量逐渐增加，表明DNA复制存在缺陷。下拉实验显示，突变破坏了GINS复合物的形成。染色质免疫沉淀实验表明，Psf2、Cut5和Cdc45的起始加载需要GINS的完整性，而Sld3则不需要。相比之下，《Psf2》在原点上的加载依赖于Sld3和Cut5，而不是Cdc45。这些结果表明，Sld3在起始复合物组装中作用最上游，其次是GINS和Cut5，最后是Cdc45。与此结论一致的是，Sld3的装载需要Cdc7-Dbf4激酶（DDK），而不需要周期蛋白依赖性激酶（C…More DK），而其他因子的募集依赖于这两种激酶。这些结果表明，在裂变酵母中，DDK和CDK调控着不同的复制起始激活步骤。真核生物染色体的DNA复制起始于一些离散的位点，称为复制起点。起源的分布和调控对于基因组的完全复制是重要的。在此，我们利用高分辨率的切片阵列技术确定了分裂糖酵母（Schizosaccharomyces pombe）全基因组中复制前复合体（pre-RC）的组成部分Orcl和Mcm6的位置。在460个基因间区中发现了Orcl和Mcm6共定位的Pre-RC位点。通过定位5-溴-2'-脱氧尿苷（BrdU）在羟基脲（HU）存在下的DNA，确定了307个pre-RC位点为早发起源。相比之下，153个未纳入BrdU的pre-RC位点被认为是延迟和/或低效的起源。Cds1缺失导致复制检查点失活，导致BrdU特异性地在起源较晚时与HU结合。早起源和晚起源往往分别分布在大的染色体区域。有趣的是，在HU存在的情况下，周围着丝粒异染色质和沉默交配型位点复制，而内部着丝粒或亚端粒异染色质则没有复制。值得注意的是，MCM不与ORC所在的内部着丝粒结合。因此，复制在染色体区域中受到不同的调控。少

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 … More 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