Pre-replication complex proteins assemble at regions of low nucleosome occupancy within the Chinese hamster dihydrofolate reductase initiation zone
Pre-replication complex proteins assemble at regions of low nucleosome occupancy within the Chinese hamster dihydrofolate reductase initiation zone
复制标题
DOI：
10.1093/nar/gkq1276
复制
发表时间：
2011-04-01
影响因子：
14.9
通讯作者：
Gilbert, David M.
中科院分区：
文献类型：
--
作者：
Lubelsky, Yoav;Sasaki, Takayo;Gilbert, David M.
哺乳动物细胞中复制前复合体蛋白的基因组规模图谱尚未有报道。这些蛋白质在特定位点富集不良可能是由于分散的结合、表位可用性差或细胞周期阶段特异性结合所致。在这里，我们使用生物素化染色质的亲和素亲和纯化，然后对 DHFR 基因座进行高密度微阵列分析，绘制了 G1 同步的中国仓鼠细胞群体中生物素标记的 ORC 和 MCM 蛋白结合位点，这些细胞含有扩增的二氢叶酸还原酶 (DHFR) 位点。我们已经确定了分布在先前确定的起始区中的这两种复合物的几个显着富集位点。对来自 DHFR 位点的拉伸 DNA 纤维的起始频率的分析证实了 ORC 和 MCM 富集位点周围存在广泛的离域起始活性区域。根据经验映射单核小体 DNA 的位置并在计算机中计算核小体定位信息表明，ORC 和 MCM 映射到测量和预测的核小体占用率较低的区域。我们的结果表明，ORC 和 MCM 富集的特定位点可以在哺乳动物起始区内检测到，并表明起始区可能是核小体占用率普遍较低的区域，其中灵活的核小体定位允许灵活的预 RC 组装位点。
Genome-scale mapping of pre-replication complex proteins has not been reported in mammalian cells. Poor enrichment of these proteins at specific sites may be due to dispersed binding, poor epitope availability or cell cycle stage-specific binding. Here, we have mapped sites of biotin-tagged ORC and MCM protein binding in G1-synchronized populations of Chinese hamster cells harboring amplified copies of the dihydrofolate reductase (DHFR) locus, using avidin-affinity purification of biotinylated chromatin followed by high-density microarray analysis across the DHFR locus. We have identified several sites of significant enrichment for both complexes distributed throughout the previously identified initiation zone. Analysis of the frequency of initiations across stretched DNA fibers from the DHFR locus confirmed a broad zone of de-localized initiation activity surrounding the sites of ORC and MCM enrichment. Mapping positions of mononucleosomal DNA empirically and computing nucleosome-positioning information in silico revealed that ORC and MCM map to regions of low measured and predicted nucleosome occupancy. Our results demonstrate that specific sites of ORC and MCM enrichment can be detected within a mammalian intitiation zone, and suggest that initiation zones may be regions of generally low nucleosome occupancy where flexible nucleosome positioning permits flexible pre-RC assembly sites.