title: A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2-7 onto DNA.
abstract: The regulated loading of the replicative helicase minichromosome maintenance proteins 2-7 (MCM2-7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2-7 hexamers into one double hexamer around dsDNA. Although the MCM2-7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. Here, we establish that the Saccharomyces cerevisiae MCM2-7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, we show that ORC-Cdc6-Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, we demonstrate that the MCM2-7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2-7 complex formation prior to MCM2-7 double hexamer formation. Our study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation.
authors.affiliation: DNA Replication Group, Institute of Clinical Science, Imperial College, London W12 0NN, United Kingdom;
authors.forename: Stefan A
authors.initials: SA
authors.lastname: Samel
authors.affiliation: DNA Replication Group, Institute of Clinical Science, Imperial College, London W12 0NN, United Kingdom;
authors.forename: Alejandra
authors.initials: A
authors.lastname: Fernández-Cid
authors.affiliation: Biosciences Department, Brookhaven National Laboratory, Upton, New York 11973, USA;
authors.forename: Jingchuan
authors.initials: J
authors.lastname: Sun
authors.affiliation: DNA Replication Group, Institute of Clinical Science, Imperial College, London W12 0NN, United Kingdom;
authors.forename: Alberto
authors.initials: A
authors.lastname: Riera
authors.affiliation: DNA Replication Group, Institute of Clinical Science, Imperial College, London W12 0NN, United Kingdom;
authors.forename: Silvia
authors.initials: S
authors.lastname: Tognetti
authors.affiliation: DNA Replication Group, Institute of Clinical Science, Imperial College, London W12 0NN, United Kingdom;
authors.forename: M Carmen
authors.initials: MC
authors.lastname: Herrera
authors.affiliation: Biosciences Department, Brookhaven National Laboratory, Upton, New York 11973, USA; Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794, USA.
authors.forename: Huilin
authors.initials: H
authors.lastname: Li
authors.affiliation: DNA Replication Group, Institute of Clinical Science, Imperial College, London W12 0NN, United Kingdom; chris.speck@imperial.ac.uk.
authors.forename: Christian
authors.initials: C
authors.lastname: Speck
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002453:Cell Cycle; D015825:Chromosomes, Fungal / Q000378:metabolism; D004271:DNA, Fungal / Q000378:metabolism*; D004789:Enzyme Activation; D006868:Hydrolysis; D064110:Minichromosome Maintenance Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018741:Replication Origin / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
chemical_list: D004271:DNA, Fungal; D046912:Multiprotein Complexes; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D064110:Minichromosome Maintenance Proteins
keywords: DNA licensing; DNA replication; cancer; genomic stability; pre-RC; replicative helicase
references.citation: Nat Struct Mol Biol. 2011 Apr;18(4):471-7
references.pmid: 21378962
references.citation: Cell. 2006 Nov 3;127(3):523-37
references.pmid: 17081975
references.citation: Genetics. 2008 Aug;179(4):1757-68
references.pmid: 18660534
references.citation: Nat Struct Mol Biol. 2014 Jan;21(1):20-5
references.pmid: 24389553
references.citation: J Biol Chem. 2010 Mar 26;285(13):9437-9443
references.pmid: 20097898
references.citation: Proc Natl Acad Sci U S A. 2008 Dec 23;105(51):20191-6
references.pmid: 19073923
references.citation: Chromosoma. 2010 Dec;119(6):565-74
references.pmid: 20686781
references.citation: Nucleic Acids Res. 2013 Mar 1;41(5):3162-72
references.pmid: 23376927
references.citation: Proc Natl Acad Sci U S A. 2012 Jul 24;109(30):11999-2004
references.pmid: 22778422
references.citation: Nat Struct Mol Biol. 2013 Aug;20(8):944-51
references.pmid: 23851460
references.citation: Cell. 2011 Jul 8;146(1):80-91
references.pmid: 21729781
references.citation: Nature. 2010 Jan 7;463(7277):113-7
references.pmid: 20054399
references.citation: Nat Rev Cancer. 2008 Oct;8(10):799-806
references.pmid: 18756287
references.citation: Nature. 2013 Mar 21;495(7441):339-43
references.pmid: 23474987
references.citation: Nature. 2007 Jan 18;445(7125):281-5
references.pmid: 17167417
references.citation: Microbiol Mol Biol Rev. 2009 Dec;73(4):652-83
references.pmid: 19946136
references.citation: Nat Cell Biol. 2006 Apr;8(4):358-66
references.pmid: 16531994
references.citation: Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):441-6
references.pmid: 9892652
references.citation: EMBO Rep. 2003 Nov;4(11):1079-83
references.pmid: 14566326
references.citation: Mol Cell. 2006 Jan 6;21(1):29-39
references.pmid: 16387651
references.citation: Proc Natl Acad Sci U S A. 2009 Dec 1;106(48):20240-5
references.pmid: 19910535
references.citation: Yeast. 1998 Jul;14(10):953-61
references.pmid: 9717241
references.citation: J Biol Chem. 2003 Feb 14;278(7):4491-9
references.pmid: 12480933
references.citation: J Biol Chem. 2011 Apr 1;286(13):11855-64
references.pmid: 21282109
references.citation: J Biol Chem. 2007 Apr 20;282(16):11705-14
references.pmid: 17314092
references.citation: Biochemistry. 2009 Mar 24;48(11):2330-9
references.pmid: 19243117
references.citation: Structure. 2012 Mar 7;20(3):534-44
references.pmid: 22405012
references.citation: Nature. 2007 Jan 18;445(7125):328-32
references.pmid: 17167415
references.citation: Cell. 2012 Oct 12;151(2):267-77
references.pmid: 23022319
references.citation: J Biol Chem. 2006 Nov 17;281(46):35531-43
references.pmid: 16980295
references.citation: Nucleic Acids Res. 2010 Oct;38(18):6078-88
references.pmid: 20484375
references.citation: Nature. 1992 May 14;357(6374):128-34
references.pmid: 1579162
references.citation: Cell. 2009 Nov 13;139(4):719-30
references.pmid: 19896182
references.citation: Semin Cell Dev Biol. 2014 Jun;30:104-9
references.pmid: 24637008
references.citation: Mol Cell. 2004 Dec 22;16(6):967-78
references.pmid: 15610739
references.citation: Cell. 2011 Sep 16;146(6):931-41
references.pmid: 21925316
references.citation: Nat Struct Biol. 2003 Mar;10(3):160-7
references.pmid: 12548282
references.citation: Cell. 2013 Apr 11;153(2):438-48
references.pmid: 23562643
references.citation: Cold Spring Harb Perspect Biol. 2013 Jul 01;5(7):
references.pmid: 23818497
references.citation: J Biol Chem. 2007 Nov 16;282(46):33795-33804
references.pmid: 17895243
references.citation: Mol Cell. 2008 Jul 25;31(2):287-93
references.pmid: 18657510
references.citation: Mol Cell Biol. 2008 Oct;28(19):5865-73
references.pmid: 18662997
references.citation: Cell. 1997 Feb 21;88(4):493-502
references.pmid: 9038340
references.citation: Cell. 1996 Oct 4;87(1):53-63
references.pmid: 8858148
references.citation: Mol Cell. 2010 Jan 29;37(2):247-58
references.pmid: 20122406
references.citation: Nat Struct Mol Biol. 2005 Nov;12(11):965-71
references.pmid: 16228006
references.citation: Mol Cell. 2013 May 23;50(4):577-88
references.pmid: 23603117
references.citation: Cell. 2000 Oct 27;103(3):375-86
references.pmid: 11081625
references.citation: Mol Cell Biol. 2007 Nov;27(21):7594-602
references.pmid: 17724082
references.citation: Virology. 1991 Sep;184(1):399-403
references.pmid: 1714665
references.citation: Proc Natl Acad Sci U S A. 1997 May 27;94(11):5611-6
references.pmid: 9159120
references.citation: EMBO J. 2011 Nov 01;30(24):4885-96
references.pmid: 22045335
references.citation: Nucleic Acids Res. 2014 Feb;42(4):2257-69
references.pmid: 24234446
references.citation: J Biol Chem. 2001 Sep 14;276(37):34768-75
references.pmid: 11432853