A helicase-tethered ORC flip enables bidirectional helicase loading.
A helicase-tethered ORC flip enables bidirectional helicase loading.
复制标题
DOI：
10.7554/elife.74282
复制
发表时间：
2021-12-09
期刊：
影响因子：
7.7
通讯作者：
Bell SP
中科院分区：
文献类型：
--
作者：
Gupta S;Friedman LJ;Gelles J;Bell SP
复制起点是通过在DNA周围以头对头的构象装载两个McM2-7解旋酶来获得许可的，以启动双向复制。这一过程需要原产地识别复合体(ORC)、CDC6和CDT1。尽管不同的CDC6和CDT1分子负载了每个解旋酶，但是否需要两个ORC蛋白尚不清楚。利用共定位单分子光谱结合单分子Förster共振能量转移(FRET)，我们研究了解旋酶负载过程中ORC与McM2-7之间的相互作用。在大多数事件中，我们观察到单个ORC分子通过类似的相互作用招募McM2-7/CDT1复合体，这些相互作用在CDT1释放后结束。在第一和第二解旋酶募集之间，ORC和第一McM2-7之间的相互作用发生了快速变化。在几秒钟内，ORC打破了调解第一个McM2-7招募的相互作用，从它最初的DNA结合部位释放出来，并与第一个McM2-7的对立面形成了新的相互作用。这种重排需要释放第一个CDT1和系链ORC，因为它翻转第一个McM2-7形成第二解旋酶募集所需的倒置McM2-7-ORC-DNA复合体。为了确保正确的许可，这个复合体一直保持到两个解旋酶之间形成头对头的相互作用。我们的发现与之前的观察结果相一致，并揭示了一系列高度协调的事件，通过这些事件，一个ORC分子可以加载两个相反方向的解旋酶。
Replication origins are licensed by loading two Mcm2-7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires origin–recognition complex (ORC), Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with single-molecule Förster resonance energy transfer (FRET), we investigated interactions between ORC and Mcm2-7 during helicase loading. In the large majority of events, we observed a single ORC molecule recruiting both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between first- and second-helicase recruitment, a rapid change in interactions between ORC and the first Mcm2-7 occurs. Within seconds, ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2-7–ORC–DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly coordinated series of events through which a single ORC molecule can load two oppositely oriented helicases.