Regulation of chromosome DNA replication for genome integrity.

真核生物中染色体DNA复制的启动是通过复制起始处的复制因子组装而受到严格调控的。我们在这里使用温度敏感的分裂酵母突变体研究了起始复合体的组装对特定因素的依赖性。突变体psf3-1是一个gins组分突变体，在限制性温度下与未复制的DNA发生滞留，DNA含量逐渐增加，表明DNA复制存在缺陷。下拉实验表明，突变会损害GINS复合体的形成。染色质免疫沉淀分析表明，PSF2、Cut5和CDC45的起始装载需要Gins的完整性，但不需要SLD3。相反，PSF2到原点的装载取决于SLD3和Cut5，而不是CDC45。这些结果表明，SLD3在起始复合体组装中的上游功能最强，其次是GINS和Cut5，然后是CDC45。与这一结论一致的是，CDC7-Dbf4激酶(Ddk)而不是细胞周期蛋白依赖性激酶(C…负载SLD3需要更多的DK)，而其他因子的募集取决于这两种激酶。这些结果表明，DDK和CDK调节分裂酵母中复制起点激活的不同步骤。真核染色体的DNA复制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很重要。在这里，我们使用高分辨率平铺阵列确定了前复制复合体(Pre-RC)的组成部分ORCL和Mcm6在裂殖酵母全基因组上的位置。在ORCL和Mcm6共定位的460个基因间隔区中发现了前RC位点。通过5-溴-2‘-脱氧尿苷(BrdU)在羟基脲(HU)存在下掺入的DNA图谱，307个RC前部位被鉴定为早期激发起源。相比之下，153个没有纳入BrdU的前RC站点被认为是较晚和/或无效的起源。CDS1缺失导致复制检查点失活，导致BrdU与HU在起始点较晚时特异地整合。早起源和晚起源倾向于分开分布在大的染色体区域。有趣的是，着丝粒周围的异染色质和沉默的交配型基因座在HU的存在下复制，而内着丝粒或亚端粒异染色质不复制。值得注意的是，MCM不与ORC所在的内部着丝粒结合。因此，复制在染色体结构域中受到不同的调节。较少

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