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Identification of New Human Origins of DNA Replication by an Origin-Trapping Assay

机译：通过原点捕获法鉴定DNA复制的新人类原点

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Metazoan genomes contain thousands of replication origins, but only a limited number have been characterized so far. We developed a two-step origin-trapping assay in which human chromatin fragments associated with origin recognition complex (ORC) in vivo were first enriched by chromatin immunoprecipitation. In a second step, these fragments were screened for transient replication competence in a plasmid-based assay utilizing the Epstein-Barr virus latent origin oriP. oriP contains two elements, an origin (dyad symmetry element [DS]) and the family of repeats, that when associated with the viral protein EBNA1 facilitate extrachromosomal stability. Insertion of the ORC-binding human DNA fragments in oriP plasmids in place of DS enabled us to screen functionally for their abilities to restore replication. Using the origin-trapping assay, we isolated and characterized five previously unknown human origins. The assay was validated with nascent strand abundance assays that confirm these origins as active initiation sites in their native chromosomal contexts. Furthermore, ORC and MCM2-7 components localized at these origins during G1 phase of the cell cycle but were not detected during mitosis. This finding extends the current understanding of origin-ORC dynamics by suggesting that replication origins must be reestablished during the early stages of each cell division cycle and that ORC itself participates in this process.
机译：后生动物基因组包含成千上万个复制起点，但到目前为止，仅有少数特征。我们开发了一个两步的来源捕获分析，其中首先通过染色质免疫沉淀富集了与体内来源识别复合物（ORC）相关的人染色质片段。第二步，利用Epstein-Barr病毒潜伏来源 oriP，在基于质粒的分析中筛选这些片段的瞬时复制能力。 oriP 包含两个元素，一个起源（双重对称元素[DS]）和重复家族，当与病毒蛋白EBNA1结合时，它们有助于染色体外稳定性。在ORM结合人DNA片段中插入代替 DS的 oriP 质粒使我们能够从功能上筛选其恢复复制的能力。使用起源捕获分析，我们分离并表征了五个以前未知的人类起源。用新生链丰度测定法验证了该测定法，该测定法证实了这些起源在其天然染色体环境中是活性起始位点。此外，ORC和MCM2-7组件在细胞周期的G 1 阶段位于这些起点，但在有丝分裂期间未检测到。这一发现通过建议必须在每个细胞分裂周期的早期重新建立复制起点，并且ORC本身参与了这一过程，从而扩展了对起源ORC动力学的当前理解。

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