Sanuki Yosuke, Kubota Yumiko, Kanemaki Masato T, Takahashi Tatsuro S, Mimura Satoru, Takisawa Haruhiko
a Department of Biological Sciences; Graduate School of Science ; Osaka University ; Toyonaka , Osaka , Japan.
Cell Cycle. 2015;14(7):1010-23. doi: 10.1080/15384101.2015.1007003.
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, using in vitro replication systems derived from Xenopus egg 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.
真核生物的DNA复制是通过进化上保守的复制蛋白逐步组装到复制起点上启动的,但在组装过程中起始DNA是如何解旋的仍不清楚。在这里,我们利用非洲爪蟾卵提取物衍生的体外复制系统,在质粒DNA上建立了一个位点特异性起点。我们发现,在缺失Cdc6的提取物中,取决于与GAL4 DNA结合结构域融合的Cdc6的结合,预复制复合物(pre-RC)优先在质粒的GAL4 DNA结合位点附近组装。随后将核质S期提取物添加到依赖GAL4的pre-RC中,促进了从起点开始的DNA复制起始,并且预起始复合物(pre-IC)和复制体的组分在起点解旋的同时被招募到起点。在这个复制系统中,RecQ4对于将Cdc45招募到起点以及Cdc45和GINS稳定结合到组装有pre-RC的质粒上都是可有可无的。然而,当从提取物中去除RecQ4时,DNA聚合酶α与起点的结合以及DNA的解旋都减少了。这些结果表明,RecQ4在脊椎动物中pre-ICs转化为需要起始DNA解旋的活性复制体的过程中起着重要作用。