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研究揭示DNA复制源头的调控机制
2022-06-19 12:09

英国弗朗西斯·克里克研究所Alessandro Costa小组揭示DNA复制源点的调控机制。相关论文于2022年6月15日在线发表在《自然》杂志上。

据研究人员介绍,真核生物复制起源的激活发生在时间上分离的步骤中,从而确保染色体在每个细胞周期只被复制一次。首先,MCM螺旋酶以非活性双六聚体的形式被加载到双联DNA上。激活发生在招募一组启动因子之后,这些因子组装了两个Cdc45-MCM-GINS(CMG)全螺旋酶。CMG的形成导致DNA在建立复制叉的过程中出现下绕,但DNA是否在这个阶段变得融化还不清楚。
 
通过用纯化的酵母蛋白在体外重新构建,研究人员用冷冻电镜观察了ATP依赖的CMG在染色质化源头上的组装。研究人员发现,CMG的形成破坏了双六聚体的界面,从而使两个CMG之间的双链DNA暴露。两个螺旋酶仍然被拴住,这就产生了一个伸展的二聚体,对源头激活和复制体的完整性有影响。在每个MCM环内,双螺旋变得不缠绕,碱基配对被破坏。这是ATP触发的MCM构象变化的结果,涉及DNA拉伸和蛋白质介导的三个孤儿碱基的稳定。在这个结构中,与DNA结合的Mcm2孔环残基对于双六聚体的装载和CMG的形成是可有可无的,但对于解除DNA的缠绕和促进复制是必不可少的。这些结果解释了ATP结合如何通过CMG使DNA成核并在起始阶段维持复制体的稳定性。
 
附:英文原文
 
Title: Mechanism of replication origin melting nucleated by CMG helicase assembly

Author: Lewis, Jacob S., Gross, Marta H., Sousa, Joana, Henrikus, Sarah S., Greiwe, Julia F., Nans, Andrea, Diffley, John F. X., Costa, Alessandro

Issue&Volume: 2022-06-15

Abstract: The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45–MCM–GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown1. Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation.

DOI: 10.1038/s41586-022-04829-4

Source: https://www.nature.com/articles/s41586-022-04829-4

Nature:《自然》,创刊于1869年。隶属于施普林格·自然出版集团,最新IF:69.504
官方网址:http://www.nature.com/
投稿链接:http://www.nature.com/authors/submit_manuscript.html


本期文章:《自然》:Online/在线发表

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