Chromosome Replication Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
Macromolecular Machines Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
Nature. 2018 Mar 8;555(7695):265-268. doi: 10.1038/nature25787. Epub 2018 Feb 28.
The initiation of eukaryotic DNA replication occurs in two discrete stages: first, the minichromosome maintenance (MCM) complex assembles as a head-to-head double hexamer that encircles duplex replication origin DNA during G1 phase; then, 'firing factors' convert each double hexamer into two active Cdc45-MCM-GINS helicases (CMG) during S phase. This second stage requires separation of the two origin DNA strands and remodelling of the double hexamer so that each MCM hexamer encircles a single DNA strand. Here we show that the MCM complex, which hydrolyses ATP during double-hexamer formation, remains stably bound to ADP in the double hexamer. Firing factors trigger ADP release, and subsequent ATP binding promotes stable CMG assembly. CMG assembly is accompanied by initial DNA untwisting and separation of the double hexamer into two discrete but inactive CMG helicases. Mcm10, together with ATP hydrolysis, then triggers further DNA untwisting and helicase activation. After activation, the two CMG helicases translocate in an 'N terminus-first' direction, and in doing so pass each other within the origin; this requires that each helicase is bound entirely to single-stranded DNA. Our experiments elucidate the mechanism of eukaryotic replicative helicase activation, which we propose provides a fail-safe mechanism for bidirectional replisome establishment.
真核生物 DNA 复制的起始发生在两个不同的阶段：首先，在 G1 期，微染色体维持（MCM）复合物作为一个首尾相接的双六聚体组装，环绕双链复制起始 DNA；然后，“点火因子”在 S 期将每个双六聚体转化为两个活性的 Cdc45-MCM-GINS 解旋酶（CMG）。第二阶段需要分离两个起始 DNA 链，并重塑双六聚体，使每个 MCM 六聚体环绕单链 DNA。在这里，我们表明，在双六聚体形成过程中水解 ATP 的 MCM 复合物在双六聚体中稳定结合 ADP。点火因子触发 ADP 释放，随后 ATP 结合促进稳定的 CMG 组装。CMG 组装伴随着初始 DNA 解旋和双六聚体分离成两个离散但非活性的 CMG 解旋酶。Mcm10 与 ATP 水解一起，然后触发进一步的 DNA 解旋和螺旋酶激活。激活后，两个 CMG 解旋酶以“N 端优先”的方向迁移，在此过程中在起始点内相互传递；这要求每个解旋酶完全结合单链 DNA。我们的实验阐明了真核复制解旋酶激活的机制，我们提出该机制为双向复制体建立提供了一种故障安全机制。
