Identification of 1600 replication origins in S. cerevisiae.
Identification of 1600 replication origins in S. cerevisiae.
复制标题
酿酒酵母 1600 个复制起点的鉴定。
DOI：
10.1101/2023.04.11.536402
复制
发表时间：
2023
期刊：
影响因子：
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通讯作者：
Bedalov,Antonio
中科院分区：
文献类型：
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作者：
Foss,EricJ;Lichauco,Carmina;Gatbonton-Schwager,Tonibelle;Gonske,SaraJ;Lofts,Brandon;Lao,Uyen;Bedalov,Antonio
在酵母基因组中有大约500个已知的复制起点，并且DNA复制在这些位置启动的过程是很好理解的。特别地，这些位点通过在S期开始之前加载Mcm复制解旋酶而能够启动复制;因此，“在G1中结合Mcm的位点”可以被认为提供了复制起点的操作定义。通过将Mcm的亚基融合到微球菌核酸酶，我们先前表明，已知的起源通常由单个Mcm双六聚体结合，加载邻近ARS共有序列（ACS）。在这里，我们将这种分析从已知的起源扩展到整个基因组，确定候选Mcm结合位点，其信号强度变化至少三个数量级。已发表的数据定量单链DNA（ssDNA）在S期的复制起始之间的最丰富的1600这些网站，与复制活动减少与Mcm丰度和消失在检测限的ssDNA。在最丰富的5500个位点中，复制起点的其他三个标志也很明显。具体而言，这些位点：（1）出现在基因间无核小体区域，一侧或两侧为定位良好的核小体;（2）两侧为ACS;（3）表现出复制起始的GC偏斜特征。我们的结论是，如果网站在Mcm双六聚体加载可以作为复制起点，那么DNA复制起点是至少三倍更丰富的比以前假设的，我们建议，复制可能偶尔开始在基本上每一个基因间区域。这些结果揭示了最近的报道，多达15%的复制事件启动以外的已知的起源，这更广泛的复制起点的分布表明，在酵母中的S期可能是不太明显的，在人类比广泛假设。
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 that binds Mcm in G1’might be considered to provide an operational definition of a replication origin. By fusing a subunit of Mcm to micrococcal nuclease, 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 three orders of magnitude. Published data quantifying single-stranded DNA (ssDNA) during S phase revealed replication initiation among the most abundant 1600 of these sites, with replication activity decreasing with Mcm abundance and disappearing at the limit of detection of ssDNA. Three other hallmarks of replication origins were apparent among the most abundant 5500 sites. Specifically, these sites:(1) appeared in intergenic nucleosome-free regions 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. We conclude that, if sites at which Mcm double hexamers are loaded can function as replication origins, then DNA replication origins are at least threefold 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 widely assumed.