title: Multiple functions for Mcm2-7 ATPase motifs during replication initiation.
abstract: The Mcm2-7 replicative helicase is central to all steps of eukaryotic DNA replication. The hexameric ring of Mcm subunits forms six essential ATPases whose contributions to replication initiation remain unclear. Mcm2-7 complexes containing ATPase-motif mutations showed Mcm2-7 ATP binding and hydrolysis are required for helicase loading. Loading-defective Mcm2-7 mutant complexes were defective in initial Mcm2-7 recruitment or Cdt1 release. Comparison with Cdc6 ATPase mutants showed that Cdc6 ATP hydrolysis is not required for helicase loading but instead drives removal of Mcm2-7 complexes that cannot complete loading. A subset of Mcm2-7 ATPase-site mutants completed helicase loading but could not initiate replication. Individual mutants were defective in distinct events during helicase activation, including maintenance of DNA association, recruitment of the GINS helicase activator, and DNA unwinding. Consistent with its heterohexameric structure, our findings show that the six Mcm2-7 ATPase active sites are specialized for different functions during helicase loading and activation.
authors.affiliation: Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Sukhyun
authors.initials: S
authors.lastname: Kang
authors.affiliation: Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Megan D
authors.initials: MD
authors.lastname: Warner
authors.affiliation: Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: spbell@mit.edu.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Bell
mesh_terms: D020816:Amino Acid Motifs; D004261:DNA Replication / Q000502:physiology*; D064110:Minichromosome Maintenance Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008957:Models, Genetic; D018741:Replication Origin
chemical_list: D064110:Minichromosome Maintenance Proteins
references.citation: Nat Struct Mol Biol. 2011 Apr;18(4):471-7
references.pmid: 21378962
references.citation: Microbiol Mol Biol Rev. 2009 Dec;73(4):652-83
references.pmid: 19946136
references.citation: Curr Biol. 2013 Apr 8;23(7):543-52
references.pmid: 23499531
references.citation: Proc Natl Acad Sci U S A. 2009 Dec 1;106(48):20240-5
references.pmid: 19910535
references.citation: J Biol Chem. 2007 Apr 20;282(16):11705-14
references.pmid: 17314092
references.citation: Nature. 2007 Jan 18;445(7125):328-32
references.pmid: 17167415
references.citation: Proc Natl Acad Sci U S A. 2006 Jul 5;103(27):10236-10241
references.pmid: 16798881
references.citation: Cell. 1997 Oct 3;91(1):59-69
references.pmid: 9335335
references.citation: Mol Cell. 1998 Jul;2(1):23-32
references.pmid: 9702188
references.citation: J Biol Chem. 2010 Mar 26;285(13):9437-9443
references.pmid: 20097898
references.citation: EMBO J. 2005 Dec 21;24(24):4334-44
references.pmid: 16369567
references.citation: Mol Cell. 2008 Jul 25;31(2):287-93
references.pmid: 18657510
references.citation: J Cell Sci. 2012 Jan 1;125(Pt 1):209-19
references.pmid: 22250202
references.citation: J Mol Biol. 2001 May 11;308(4):597-608
references.pmid: 11350163
references.citation: Nucleic Acids Res. 2013 Mar 1;41(5):3162-72
references.pmid: 23376927
references.citation: Cell. 2011 Jul 8;146(1):80-91
references.pmid: 21729781
references.citation: Genes Dev. 2010 Mar 15;24(6):602-12
references.pmid: 20231317
references.citation: Mol Cell. 2001 Nov;8(5):1093-104
references.pmid: 11741544
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: Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):441-6
references.pmid: 9892652
references.citation: Mol Cell. 2006 Jan 6;21(1):29-39
references.pmid: 16387651
references.citation: Genes Dev. 2009 Mar 1;23(5):643-54
references.pmid: 19270162
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: Nat Cell Biol. 2002 Mar;4(3):198-207
references.pmid: 11836525
references.citation: Nat Struct Mol Biol. 2014 Jan;21(1):20-5
references.pmid: 24389553
references.citation: Mol Cell Biol. 2013 Jul;33(13):2614-22
references.pmid: 23629628
references.citation: Mol Cell. 2004 Dec 22;16(6):967-78
references.pmid: 15610739
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: Cold Spring Harb Perspect Biol. 2013 Dec 01;5(12):a010371
references.pmid: 23881938
references.citation: Curr Biol. 2011 Dec 20;21(24):2055-63
references.pmid: 22169533
references.citation: Trends Biochem Sci. 2013 Apr;38(4):184-94
references.pmid: 23332289
references.citation: Cold Spring Harb Perspect Biol. 2013 Apr 01;5(4):a010165
references.pmid: 23545418
references.citation: Nat Struct Mol Biol. 2013 Aug;20(8):944-51
references.pmid: 23851460
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: Annu Rev Biochem. 2013;82:25-54
references.pmid: 23746253
references.citation: Proc Natl Acad Sci U S A. 1997 May 27;94(11):5611-6
references.pmid: 9159120