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Nat Struct Mol Biol. 2017 Mar;24(3):309-315. doi: 10.1038/nsmb.3375. Epub 2017 Feb 13.

Mechanism and timing of Mcm2-7 ring closure during DNA replication origin licensing.

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Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Department of Biochemistry, Brandeis University, Waltham, Massachusetts, USA.
New England BioLabs, Ipswich, Massachusetts, USA.


The opening and closing of two ring-shaped Mcm2-7 DNA helicases is necessary to license eukaryotic origins of replication, although the mechanisms controlling these events are unclear. The origin-recognition complex (ORC), Cdc6 and Cdt1 facilitate this process by establishing a topological link between each Mcm2-7 hexamer and origin DNA. Using colocalization single-molecule spectroscopy and single-molecule Förster resonance energy transfer (FRET), we monitored ring opening and closing of Saccharomyces cerevisiae Mcm2-7 during origin licensing. The two Mcm2-7 rings were open during initial DNA association and closed sequentially, concomitant with the release of their associated Cdt1. We observed that ATP hydrolysis by Mcm2-7 was coupled to ring closure and Cdt1 release, and failure to load the first Mcm2-7 prevented recruitment of the second Mcm2-7. Our findings identify key mechanisms controlling the Mcm2-7 DNA-entry gate during origin licensing, and reveal that the two Mcm2-7 complexes are loaded via a coordinated series of events with implications for bidirectional replication initiation and quality control.

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