Identification of 1600 replication origins in S. cerevisiae

There are approximately 500 known origins of replication in the yeast genome, and the process by which DNA replication initiates at these locations is well understood. In particular, these sites are made competent to initiate replication by loading of the Mcm replicative helicase prior to the start of S phase; thus, a site to which MCM is bound in G1 might be considered to provide an operational definition of a replication origin. By fusing a subunit of Mcm to micrococcal nuclease, a technique referred to as Chromatin Endogenous Cleavage, we previously showed that known origins are typically bound by a single Mcm double hexamer, loaded adjacent to the ARS consensus sequence (ACS). Here we extend this analysis from known origins to the entire genome, identifying candidate Mcm binding sites whose signal intensity varies over at least 3 orders of magnitude. Published data quantifying the production of ssDNA during S phase showed clear evidence of replication initiation among the most abundant 1600 of these sites, with replication activity decreasing in concert with Mcm abundance and disappearing at the limit of detection of ssDNA. Three other hallmarks of replication origins were apparent among the most abundant 5,500 sites. Specifically, these sites (1) appeared in intergenic nucleosome-free regions that were flanked on one or both sides by well-positioned nucleosomes; (2) were flanked by ACSs; and (3) exhibited a pattern of GC skew characteristic of replication initiation. Furthermore, the high resolution of this technique allowed us to demonstrate a strong bias for detecting Mcm double-hexamers downstream rather than upstream of the ACS, which is consistent with the directionality of Mcm loading by Orc that has been observed in vitro. We conclude that DNA replication origins are at least 3-fold more abundant than previously assumed, and we suggest that replication may occasionally initiate in essentially every intergenic region. These results shed light on recent reports that as many as 15% of replication events initiate outside of known origins, and this broader distribution of replication origins suggest that S phase in yeast may be less distinct from that in humans than is widely assumed.酵母基因组中已知的复制起点（replication origin）约有500个，在这些位点启动DNA复制的过程已得到充分阐明。具体而言，在S期起始前，Mcm复制解旋酶（Mcm replicative helicase）的装载会赋予这些位点复制启动的能力；因此，G1期结合有MCM的位点，可被视为复制起点的操作性定义。我们此前通过将Mcm的一个亚基与微球菌核酸酶（micrococcal nuclease）融合的技术——即染色质内源切割（Chromatin Endogenous Cleavage，ChEC）——证实，已知复制起点通常仅结合单个Mcm双六聚体，且该六聚体装载于ARS共识序列（ARS consensus sequence，ACS）的旁侧。本研究将该分析从已知复制起点拓展至全基因组范围，鉴定出候选Mcm结合位点，其信号强度跨度至少达3个数量级。已发表的S期单链DNA（single-stranded DNA，ssDNA）生成定量数据显示，在这些位点中丰度最高的1600个位点内，存在明确的复制起始证据；复制活性随Mcm丰度降低而减弱，在ssDNA的检测限下则完全消失。在丰度最高的5500个位点中，还观察到复制起点的另外三个标志性特征：其一，这些位点位于基因间区的核小体缺失区域，且单侧或双侧被定位良好的核小体所侧翼；其二，其旁侧存在ACS；其三，呈现出与复制起始特征相符的GC偏斜模式。此外，该技术的高分辨率使我们得以证实，Mcm双六聚体的检测在ACS下游存在显著偏好性，而非上游，这与体外实验中观察到的Orc介导的Mcm装载方向性一致。我们据此得出结论：酵母基因组中的DNA复制起点数量至少比此前假设的多3倍，并提出复制或许偶尔会在几乎所有基因间区起始。这些结果为近期报道的多达15%的复制事件起始于已知起点之外的现象提供了阐释，且这种更广泛的复制起点分布表明，酵母的S期与人类的S期或许并不像此前普遍认为的那样差异显著。