Pacek Marcin, Walter Johannes C
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
EMBO J. 2004 Sep 15;23(18):3667-76. doi: 10.1038/sj.emboj.7600369. Epub 2004 Aug 26.
In vertebrates, MCM2-7 and Cdc45 are required for DNA replication initiation, but it is unknown whether they are also required for elongation, as in yeast. Moreover, although MCM2-7 is a prime candidate for the eukaryotic replicative DNA helicase, a demonstration that MCM2-7 unwinds DNA during replication is lacking. Here, we use Xenopus egg extracts to investigate the roles of MCM7 and Cdc45 in DNA replication. A fragment of the retinoblastoma protein, Rb(1-400), was used to neutralize MCM7, and antibodies were used to neutralize Cdc45. When added immediately after origin unwinding, or after significant DNA synthesis, both inhibitors blocked further DNA replication, indicating that MCM7 and Cdc45 are required throughout replication elongation in vertebrates. We next exploited the fact that inhibition of DNA polymerase by aphidicolin causes extensive chromosome unwinding, likely due to uncoupling of the replicative DNA helicase. Strikingly, Rb(1-400) and Cdc45 antibodies both abolished unwinding by the uncoupled helicase. These results provide new support for the model that MCM2-7 is the replicative DNA helicase, and they indicate that Cdc45 functions as a helicase co-factor.
在脊椎动物中，DNA复制起始需要MCM2 - 7和Cdc45，但它们是否也像在酵母中一样参与延伸过程尚不清楚。此外，尽管MCM2 - 7是真核生物复制性DNA解旋酶的主要候选者，但缺乏MCM2 - 7在复制过程中解旋DNA的证据。在这里，我们利用非洲爪蟾卵提取物来研究MCM7和Cdc45在DNA复制中的作用。使用视网膜母细胞瘤蛋白的一个片段Rb(1 - 400)来中和MCM7，并使用抗体来中和Cdc45。当在起始点解旋后立即添加，或在大量DNA合成后添加时，两种抑制剂都阻断了进一步的DNA复制，这表明在脊椎动物的整个复制延伸过程中都需要MCM7和Cdc45。接下来，我们利用了放线菌素抑制DNA聚合酶会导致广泛的染色体解旋这一事实，这可能是由于复制性DNA解旋酶的解偶联所致。令人惊讶的是，Rb(1 - 400)和Cdc45抗体都消除了未偶联解旋酶引起的解旋。这些结果为MCM2 - 7是复制性DNA解旋酶的模型提供了新的支持，并表明Cdc45作为解旋酶辅助因子发挥作用。
