Unwinding of a eukaryotic origin of replication visualized by cryo-EM

IF 12.5 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Nature Structural & Molecular Biology Pub Date : 2024-05-17 DOI:10.1038/s41594-024-01280-z
Sarah S. Henrikus, Marta H. Gross, Oliver Willhoft, Thomas Pühringer, Jacob S. Lewis, Allison W. McClure, Julia F. Greiwe, Giacomo Palm, Andrea Nans, John F. X. Diffley, Alessandro Costa
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Abstract

To prevent detrimental chromosome re-replication, DNA loading of a double hexamer of the minichromosome maintenance (MCM) replicative helicase is temporally separated from DNA unwinding. Upon S-phase transition in yeast, DNA unwinding is achieved in two steps: limited opening of the double helix and topological separation of the two DNA strands. First, Cdc45, GINS and Polε engage MCM to assemble a double CMGE with two partially separated hexamers that nucleate DNA melting. In the second step, triggered by Mcm10, two CMGEs separate completely, eject the lagging-strand template and cross paths. To understand Mcm10 during helicase activation, we used biochemical reconstitution with cryogenic electron microscopy. We found that Mcm10 splits the double CMGE by engaging the N-terminal homo-dimerization face of MCM. To eject the lagging strand, DNA unwinding is started from the N-terminal side of MCM while the hexamer channel becomes too narrow to harbor duplex DNA. Here the authors used cryogenic electron microscopy and biochemistry to understand how yeast Mcm10 exerts its essential role in DNA replication initiation, finding that it splits the double Cdc45-MCM-GINS-Polε structure. The lagging-strand template is ejected from each MCM ring as the central channel of the helicase becomes too tight to accommodate two DNA strands.

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通过低温电子显微镜观察真核生物复制源的开卷过程
为了防止有害的染色体再复制,微型染色体维护(MCM)复制螺旋酶双六聚体的 DNA 加载与 DNA 解旋在时间上是分离的。在酵母的 S 期转换过程中,DNA 解旋分为两个步骤:双螺旋的有限开放和两条 DNA 链的拓扑分离。首先,Cdc45、GINS 和 Polε 与 MCM 结合,组装出带有两个部分分离的六聚体的双 CMGE,从而核化 DNA 熔化。第二步由 Mcm10 触发,两个 CMGE 完全分离,弹出滞后链模板并交叉。为了了解螺旋酶激活过程中的 Mcm10,我们使用了生化重组和低温电子显微镜。我们发现,Mcm10 通过与 MCM 的 N 端同源二聚化面接合来分裂双 CMGE。为了排出滞后链,DNA 从 MCM 的 N 端开始解旋,同时六聚体通道变得过于狭窄,无法容纳双链 DNA。
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来源期刊
Nature Structural & Molecular Biology
Nature Structural & Molecular Biology BIOCHEMISTRY & MOLECULAR BIOLOGY-BIOPHYSICS
CiteScore
22.00
自引率
1.80%
发文量
160
审稿时长
3-8 weeks
期刊介绍: Nature Structural & Molecular Biology is a comprehensive platform that combines structural and molecular research. Our journal focuses on exploring the functional and mechanistic aspects of biological processes, emphasizing how molecular components collaborate to achieve a particular function. While structural data can shed light on these insights, our publication does not require them as a prerequisite.
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