Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast
Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast
复制标题
DOI：
10.1038/sj.emboj.7601585
复制
发表时间：
2007-03-07
期刊：
影响因子：
11.4
通讯作者：
Masukata, Hisao
中科院分区：
文献类型：
--
作者：
Hayashi, Makoto;Katou, Yuki;Masukata, Hisao
真核染色体的 DNA 复制起始于许多离散的基因座，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 Orc1 和 Mcm6（预复制复合体（pre-RC）的组成部分）在粟酒裂殖酵母整个基因组上的位置。在 460 个基因间区域中鉴定出 Pre-RC 位点，其中 Orc1 和 Mcm6 共定位。通过在羟基脲 (HU) 存在下对掺入 5-溴-2'-脱氧尿苷 (BrdU) 的 DNA 进行绘图，将 307 个前 RC 位点确定为早期放电起源。相比之下，未掺入 BrdU 的 153 个 pre-RC 位点被认为是晚期和/或低效起源。 Cds1 缺失导致复制检查点失活，导致 BrdU 在晚期起源处与 HU 结合。早期和晚期起源往往分别分布在较大的染色体区域中。有趣的是，着丝粒周围异染色质和沉默交配型基因座在 HU 存在的情况下进行复制，而内部着丝粒或亚端粒异染色质则不会。值得注意的是，MCM 不与起源识别复合物所在的内部着丝粒结合。因此，复制在染色体结构域中受到差异性调节。
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 Orc1 and Mcm6, components of pre-replicative complex (pre-RC), on the whole genome of Schizosaccharomyces pombe using a high-resolution tiling array. Pre-RC sites were identified in 460 intergenic regions, where Orc1 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 earlyfiring 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, whereas the inner centromere or subtelomeric heterochromatin did not. Notably, MCM did not bind to inner centromeres where origin recognition complex was located. Thus, replication is differentially regulated in chromosome domains.