Identification of new human origins of DNA replication by an origin-trapping assay
Identification of new human origins of DNA replication by an origin-trapping assay
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DOI：
10.1128/mcb.01392-06
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发表时间：
2006-10-01
影响因子：
5.3
通讯作者：
Schepers, Aloys
中科院分区：
文献类型：
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作者：
Gerhardt, Jeannine;Jafar, Samira;Schepers, Aloys
后生动物基因组包含数千个复制起点，但到目前为止只有有限的数量被表征。我们建立了一种两步来源捕获实验，其中与体内来源识别复合体(ORC)相关的人染色质片段首先通过染色质免疫沉淀富集。在第二步中，利用Epstein-Barr病毒潜伏起源ORIP，在基于质粒的检测中筛选这些片段的瞬时复制能力。ORIP包含两个元件，一个起源(二联体对称元件[DS])和重复序列家族，当与病毒蛋白EBNA1结合时，有助于染色体外的稳定性。将ORC结合的人类DNA片段插入ORIP质粒中以取代DS，使我们能够从功能上筛选它们恢复复制的能力。利用起源捕捉实验，我们分离并鉴定了五个以前未知的人类起源。该分析通过新生的链丰度分析进行验证，证实这些来源在其天然染色体环境中是活跃的起始位点。此外，ORC和MCM2-7组分在细胞周期的G(1)期定位于这些起始点，但在有丝分裂中未检测到。这一发现扩展了目前对起源-ORC动力学的理解，提出复制起点必须在每个细胞分裂周期的早期阶段重新建立，并且ORC本身参与了这一过程。
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 G(1) 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.