Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6

Yuan, Zuanning, Schneider, Sarah, Dodd, Thomas, Riera, A., Bai, Lin, Yan, Chunli, Magdalou, Indiana, Ivanov, Ivaylo, Stillman, Bruce, Li, Huilin, Speck, C. (July 2020) Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6. Proceedings of the National Academy of Sciences of the United States of America, 117 (30). pp. 17747-17756. ISSN 0027-8424

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Abstract

DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC-Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA. A key intermediate is the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) complex in which DNA has been already inserted into the central channel of Mcm2-7. Until now, it has been unclear how the origin DNA is guided by ORC-Cdc6 and inserted into the Mcm2-7 hexamer. Here, we truncated the C-terminal winged-helix-domain (WHD) of Mcm6 to slow down the loading reaction, thereby capturing two loading intermediates prior to DNA insertion in budding yeast. In "semi-attached OCCM," the Mcm3 and Mcm7 WHDs latch onto ORC-Cdc6 while the main body of the Mcm2-7 hexamer is not connected. In "pre-insertion OCCM," the main body of Mcm2-7 docks onto ORC-Cdc6, and the origin DNA is bent and positioned adjacent to the open DNA entry gate, poised for insertion, at the Mcm2-Mcm5 interface. We used molecular simulations to reveal the dynamic transition from preloading conformers to the loaded conformers in which the loading of Mcm2-7 on DNA is complete and the DNA entry gate is fully closed. Our work provides multiple molecular insights into a key event of eukaryotic DNA replication.

Item Type:	Paper
Subjects:	Investigative techniques and equipment > microscopy > Cryo-electron microscopy bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA replication Investigative techniques and equipment organism description > yeast > Saccharomyces organs, tissues, organelles, cell types and functions > cell types and functions > cell functions > cell cycle organs, tissues, organelles, cell types and functions > cell types and functions > cell functions organs, tissues, organelles, cell types and functions > cell types and functions bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > helicase Investigative techniques and equipment > microscopy organs, tissues, organelles, cell types and functions bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > origin recognition complex organism description > yeast
CSHL Authors:	Stillman, Bruce W.
Communities:	CSHL labs > Stillman lab CSHL Cancer Center Program > Gene Regulation and Inheritance Program
Highlight:	Stillman, Bruce W.
Depositing User:	Matthew Dunn
Date:	28 July 2020
Date Deposited:	03 Dec 2020 19:45
Last Modified:	01 Feb 2024 19:47
PMCID:	PMC7395460
Related URLs:	Publisher
URI:	https://repository.cshl.edu/id/eprint/39833

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