PDF(Pubmed)
Abstract：
The chromatin environment at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how and which chromatin features control the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we use site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae. Using mass spectrometry, we define the protein composition of native chromatin regions surrounding the EE and LI replication start sites. In addition to known origin interactors, we find the microtubule-binding Ask1/DASH complex as an origin-regulating factor. Strikingly, tethering of Ask1 to individual origin sites advances replication timing (RT) of the targeted chromosomal domain. Targeted degradation of Ask1 globally changes RT of a subset of origins, which can be reproduced by inhibiting microtubule dynamics. Thus, our findings mechanistically connect RT and chromosomal organization via Ask1/DASH with the microtubule cytoskeleton.
摘要：
复制起点的染色质环境被认为影响真核基因组中的DNA复制起始。然而,目前尚不清楚染色质特征如何以及哪些特征控制早期有效(EE)或晚期无效(LI)起源的激发。这里,我们使用位点特异性重组和单基因座染色质分离纯化酿酒酵母中的EE和LI复制起点。使用质谱,我们定义了围绕EE和LI复制起始位点的天然染色质区域的蛋白质组成。除了已知的起源相互作用者，我们发现微管结合Ask1/DASH复合物是起源调节因子。引人注目的是，将Ask1连接到单个起源位点会提高目标染色体结构域的复制时机（RT）。Ask1的目标降解在全球范围内改变了起源子集的RT，可以通过抑制微管动力学来复制。因此，我们的发现通过Ask1/DASH将RT和染色体组织与微管细胞骨架进行机械连接。
