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-Structure paper
Title | Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6. |
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Journal, issue, pages | Proc Natl Acad Sci U S A, Vol. 117, Issue 30, Page 17747-17756, Year 2020 |
Publish date | Jul 28, 2020 |
Authors | Zuanning Yuan / Sarah Schneider / Thomas Dodd / Alberto Riera / Lin Bai / Chunli Yan / Indiana Magdalou / Ivaylo Ivanov / Bruce Stillman / Huilin Li / Christian Speck / |
PubMed 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- ...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. |
External links | Proc Natl Acad Sci U S A / PubMed:32669428 / PubMed Central |
Methods | EM (single particle) |
Resolution | 4.3 - 10.0 Å |
Structure data | EMDB-21662: Cryo-EM map of semi-attached mutant OCCM-DNA complex (ORC-Cdc6-Cdt1-Mcm2-7 with Mcm6 WHD truncation) EMDB-21664: Cryo-EM map of mutant Mcm2-7 hexamer with Mcm6 WHD truncation EMDB-21665: Cryo-EM map of pre-insertion mutant OCCM-DNA complex (ORC-Cdc6-Cdt1-Mcm2-7 with Mcm6 WHD truncation) EMDB-21666: Cryo-EM map of the mutant OCCM (ORC-Cdc6-Cdt1-Mcm2-7 with Mcm6 WHD truncation) loaded on DNA at 10.5 A resolution |
Chemicals | ChemComp-AGS: ChemComp-MG: |
Source |
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Keywords | REPLICATION/DNA / DNA replication / Cryo-EM / OCCM-deltaC6 / REPLICATION-DNA complex / REPLICATION |