title: Mechanism and timing of Mcm2-7 ring closure during DNA replication origin licensing.
abstract: 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.
authors.affiliation: Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Simina
authors.initials: S
authors.lastname: Ticau
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, Massachusetts, USA.
authors.forename: Larry J
authors.initials: LJ
authors.lastname: Friedman
authors.affiliation: Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Kanokwan
authors.initials: K
authors.lastname: Champasa
authors.affiliation: New England BioLabs, Ipswich, Massachusetts, USA.
authors.forename: Ivan R
authors.initials: IR
authors.lastname: Corrêa
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, Massachusetts, USA.
authors.forename: Jeff
authors.identifier: 0000-0001-7910-3421
authors.initials: J
authors.lastname: Gelles
authors.affiliation: Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Stephen P
authors.identifier: 0000-0002-2876-610X
authors.initials: SP
authors.lastname: Bell
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D004261:DNA Replication; D004271:DNA, Fungal / Q000378:metabolism; D006868:Hydrolysis; D064110:Minichromosome Maintenance Proteins / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D011487:Protein Conformation; D055503:Protein Multimerization; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013997:Time Factors
chemical_list: D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D064110:Minichromosome Maintenance Proteins