Mechanisms of MCM2-7 loading and initial DNA melting at near base-pair resolution

The loading and activation of the replicative helicase MCM2-7 are key events during the G1-S phase transition.In budding yeast, the origin recognition complex (ORC) binds to the conserved DNA elements A and B1 of the autonomously replicating sequence (ARS).This is followed by the consecutive loading of two MCM2-7 hetero-hexamers into a MCM2-7 double-hexamer(DH).In S-phase the MCM2-7DH is activated, resulting in two Cdc45-MCM-GINS(CMG) helicases that bidirectionally unwind DNA ahead of the replication fork.Here,we show that MCM2-7 helicase loading across the B1 element displaces ORC fromo rigins.This allows ORC binding and helicase loading at lower affinity binding sites and origins throughout the genome.Furthermore, we mapped the sites of initial DNA unwinding genome-wide and show that these sites appear near the N-terminal domains of the MCM2-7 double-hexamer in proximity of the B1 element.Finally, employing a chemical-biology approach, we establish that during helicase activation the Mcm2/5 interface acts as the DNA exit gate for single-stranded-DNA extrusion.Our work identifies that helicase loading follows a distributive mechanism, allowing for equal MCM2-7 loading across the genome and surprisingly finds that DNA unwinding initiates from the helicase N-terminal interface in proximity to the ARS B1 element.复制解旋酶MCM2-7（replicative helicase MCM2-7）的装载与激活是G1-S期转换过程中的关键事件。在出芽酵母中，起始识别复合物（origin recognition complex, ORC）会结合到自主复制序列（autonomously replicating sequence, ARS）上保守的DNA元件A与B1。随后，两个MCM2-7异六聚体依次装载形成MCM2-7双六聚体（double-hexamer, DH）。在S期，MCM2-7 DH被激活，产生两个Cdc45-MCM-GINS（CMG）解旋酶，在复制叉前方双向解旋DNA。本研究显示，跨B1元件的MCM2-7解旋酶装载会将ORC从复制起点处置换下来，这使得ORC能够结合基因组中亲和力较低的结合位点与复制起点，并完成解旋酶的装载。此外，我们通过全基因组定位了初始DNA解旋的位点，发现这些位点出现在靠近B1元件的MCM2-7双六聚体N端结构域附近。最后，借助化学生物学方法，我们证实：在解旋酶激活过程中，Mcm2/5界面作为单链DNA挤出的DNA出口闸门。本研究表明，解旋酶装载遵循分布式机制，可实现MCM2-7在全基因组范围内的均等装载；同时意外发现，DNA解旋起始于ARS B1元件附近的解旋酶N端界面。