RecQ4 promotes the conversion of the pre-initiation complex at a site-specific origin for DNA unwinding in Xenopus egg extracts
RecQ4 promotes the conversion of the pre-initiation complex at a site-specific origin for DNA unwinding in Xenopus egg extracts
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RecQ4 促进非洲爪蟾卵提取物中 DNA 解旋起始位点前复合物的转化
DOI：
10.1080/15384101.2015.1007003
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发表时间：
2015
期刊：
影响因子：
4.3
通讯作者：
Takisawa H
中科院分区：
文献类型：
--
作者：
Sanuki;Kubota;Kanemaki MT;Takahashi TS;Mimura S;Takisawa H
真核生物的DNA复制是通过将进化上保守的复制蛋白逐步组装到复制起点上来启动的，但在组装过程中原始DNA是如何解开的仍不清楚。在这里，我们建立了一个特定的位置起源于一个质粒DNA，使用的体外复制系统来自非洲爪哇虫卵提取液。我们发现，预复制复合体(Pre-RC)优先组装在质粒的GAL4 DNA结合部位附近，这取决于CDC6与CdC6缺失提取物中GAL4 DNA结合域的结合。随后在依赖Gal4的Pre-RC中加入核质S相提取物，促进了从起源开始的DNA复制，并在起始解离的同时将预起始复合体(PreIC)和复制体的成分招募到起源。在这个复制系统中，RecQ4对于CDC45在起始点上的重新募集和CDC45和GINS与Pre-RC组装的质粒的稳定结合都是必不可少的。然而，当提取液中的RecQ4被耗尽时，DNA聚合酶α的起始结合和DNA解离都减少了。这些结果表明，在脊椎动物中，RecQ4在将前IC转化为需要解开原始DNA的活跃复制体过程中发挥着重要作用。
Eukaryotic DNA replication is initiated through stepwise assembly of evolutionarily conserved replication proteins onto replication origins, but how the origin DNA is unwound during the assembly process remains elusive. Here, we established a site-specific origin on a plasmid DNA, usingin vitroreplication systems derived fromXenopusegg extracts. We found that the pre-replicative complex (pre-RC) was preferentially assembled in the vicinity of GAL4 DNA-binding sites of the plasmid, depending on the binding of Cdc6 fused with a GAL4 DNA-binding domain in Cdc6-depleted extracts. Subsequent addition of nucleoplasmic S-phase extracts to the GAL4-dependent pre-RC promoted initiation of DNA replication from the origin, and components of the pre-initiation complex (pre-IC) and the replisome were recruited to the origin concomitant with origin unwinding. In this replication system, RecQ4 is dispensable for both recruitment of Cdc45 onto the origin and stable binding of Cdc45 and GINS to the pre-RC assembled plasmid. However, both origin binding of DNA polymerase α and unwinding of DNA were diminished upon depletion of RecQ4 from the extracts. These results suggest that RecQ4 plays an important role in the conversion of pre-ICs into active replisomes requiring the unwinding of origin DNA in vertebrates.
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