The eukaryotic helicase MCM2-7, is loaded by ORC, Cdc6 and Cdt1 as a double-hexamer onto replication origins. The insertion of DNA into the helicase leads to partial MCM2-7 ring closure, while ATP hydrolysis is essential for consecutive steps in pre-replicative complex (pre-RC) assembly. Currently it is unknown how MCM2-7 ring closure and ATP-hydrolysis are controlled. A cryo-EM structure of an ORC-Cdc6-Cdt1-MCM2-7 intermediate shows a remodelled, fully-closed Mcm2/Mcm5 interface. The Mcm5 C-terminus (C5) contacts Orc3 and specifically recognises this closed ring. Interestingly, we found that normal helicase loading triggers Mcm4 ATP-hydrolysis, which in turn leads to reorganisation of the MCM2-7 complex and Cdt1 release. However, defective MCM2-7 ring closure, due to mutations at the Mcm2/Mcm5 interface, leads to MCM2-7 ring splitting and complex disassembly. As such we identify Mcm4 as the key ATPase in regulating pre-RC formation. Crucially, a stable Mcm2/Mcm5 interface is essential for productive ATP-hydrolysis-dependent remodelling of the helicase.
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MCM2-7 ring closure involves the Mcm5 C-terminus and triggers Mcm4 ATP hydrolysis.
Nat Commun
January 2025
DNA Replication Group, Institute of Clinical Science, Imperial College London, London, UK.
The eukaryotic helicase MCM2-7, is loaded by ORC, Cdc6 and Cdt1 as a double-hexamer onto replication origins. The insertion of DNA into the helicase leads to partial MCM2-7 ring closure, while ATP hydrolysis is essential for consecutive steps in pre-replicative complex (pre-RC) assembly. Currently it is unknown how MCM2-7 ring closure and ATP-hydrolysis are controlled.
View Article and Find Full Text PDFMultiple mechanisms for licensing human replication origins.
Nature
December 2024
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
Article Synopsis
- The loading of MCM2-7 helicases is crucial for assembling DNA replication machinery, with human loading pathways differing from that of yeast, particularly regarding the role of ORC6.
- Through biochemical reconstitution and electron microscopy, researchers identified several intermediate states in the formation of MCM double hexamers, revealing that ORC6 enhances but is not essential for loading MCM onto DNA.
- The flexibility in the human loading process, including the potential dimerization of independently loaded MCM hexamers, may offer advantages during cellular replication stress, paving the way for future studies on DNA replication initiation.
Multiple pathways for licensing human replication origins.
bioRxiv
April 2024
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
The loading of replicative helicases constitutes an obligatory step in the assembly of DNA replication machineries. In eukaryotes, the MCM2-7 replicative helicase motor is deposited onto DNA by the origin recognition complex (ORC) and co-loader proteins as a head-to-head MCM double hexamer to license replication origins. Although extensively studied in the budding yeast model system, the mechanisms of origin licensing in higher eukaryotes remain poorly defined.
View Article and Find Full Text PDFMechanism of eukaryotic origin unwinding is a dual helicase DNA shearing process.
Proc Natl Acad Sci U S A
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The Rockefeller University, New York City, NY 10065.
Article Synopsis
- DNA replication starts with separating the base pairs at the origin to access single-stranded DNA templates for replication by DNA polymerases.
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Regulation of replication origin licensing by ORC phosphorylation reveals a two-step mechanism for Mcm2-7 ring closing.
Proc Natl Acad Sci U S A
July 2023
HHMI, Massachusetts Institute of Technology, Cambridge, MA 02139.
Article Synopsis
- Eukaryotic DNA replication is tightly regulated to occur only once per cell cycle to maintain proper cell ploidy.
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