title: Overexpression of the Replicative Helicase in Escherichia coli Inhibits Replication Initiation and Replication Fork Reloading.
abstract: Replicative helicases play central roles in chromosome duplication and their assembly onto DNA is regulated via initiators and helicase loader proteins. The Escherichia coli replicative helicase DnaB and the helicase loader DnaC form a DnaB6-DnaC6 complex that is required for loading DnaB onto single-stranded DNA. Overexpression of dnaC inhibits replication by promoting continual rebinding of DnaC to DnaB and consequent prevention of helicase translocation. Here we show that overexpression of dnaB also inhibits growth and chromosome duplication. This inhibition is countered by co-overexpression of wild-type DnaC but not of a DnaC mutant that cannot interact with DnaB, indicating that a reduction in DnaB6-DnaC6 concentration is responsible for the phenotypes associated with elevated DnaB concentration. Partial defects in the oriC-specific initiator DnaA and in PriA-specific initiation away from oriC during replication repair sensitise cells to dnaB overexpression. Absence of the accessory replicative helicase Rep, resulting in increased replication blockage and thus increased reinitiation away from oriC, also exacerbates DnaB-induced defects. These findings indicate that elevated levels of helicase perturb replication initiation not only at origins of replication but also during fork repair at other sites on the chromosome. Thus, imbalances in levels of the replicative helicase and helicase loader can inhibit replication both via inhibition of DnaB6-DnaC6 complex formation with excess DnaB, as shown here, and promotion of formation of DnaB6-DnaC6 complexes with excess DnaC [Allen GC, Jr., Kornberg A. Fine balance in the regulation of DnaB helicase by DnaC protein in replication in Escherichia coli. J. Biol. Chem. 1991;266:22096-22101; Skarstad K, Wold S. The speed of the Escherichia coli fork in vivo depends on the DnaB:DnaC ratio. Mol. Microbiol. 1995;17:825-831]. Thus, there are two mechanisms by which an imbalance in the replicative helicase and its associated loader protein can inhibit genome duplication.
authors.affiliation: Department of Biology, University of York, Wentworth Way, York YO10 5DD, United Kingdom.
authors.forename: Jan-Gert
authors.initials: JG
authors.lastname: Brüning
authors.affiliation: Department of Biology, University of York, Wentworth Way, York YO10 5DD, United Kingdom.
authors.forename: Kamila Katarzyna
authors.initials: KK
authors.lastname: Myka
authors.affiliation: Department of Biology, University of York, Wentworth Way, York YO10 5DD, United Kingdom. Electronic address: peter.mcglynn@york.ac.uk.
authors.forename: Peter
authors.initials: P
authors.lastname: McGlynn
mesh_terms: D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D053488:DnaB Helicases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015870:Gene Expression
chemical_list: D004277:DNA, Single-Stranded; C071702:DnaC protein, E coli; D029968:Escherichia coli Proteins; C506730:dnaB protein, E coli; D053488:DnaB Helicases
keywords: DNA repair; DNA replication; disease; genome stability; transcription
references.citation: J Biol Chem. 2005 Dec 2;280(48):39693-700
references.pmid: 16188886
references.citation: J Biol Chem. 1996 Jun 28;271(26):15642-8
references.pmid: 8663104
references.citation: Mol Microbiol. 2006 May;60(4):973-83
references.pmid: 16677308
references.citation: Nucleic Acids Res. 2006;34(18):5194-202
references.pmid: 17000639
references.citation: Nat Rev Microbiol. 2007 May;5(5):343-54
references.pmid: 17435790
references.citation: Mol Cell. 2007 Jun 22;26(6):781-93
references.pmid: 17588514
references.citation: Mol Cell. 1999 Oct;4(4):541-53
references.pmid: 10549286
references.citation: Cell. 2000 Mar 31;101(1):35-45
references.pmid: 10778854
references.citation: Genetics. 2000 Jun;155(2):487-97
references.pmid: 10835375
references.citation: J Mol Biol. 2000 Aug 11;301(2):285-99
references.pmid: 10926510
references.citation: J Biol Chem. 2001 Jul 20;276(29):27345-53
references.pmid: 11333269
references.citation: Mol Microbiol. 2001 Aug;41(3):697-704
references.pmid: 11532137
references.citation: EMBO J. 2002 Jun 17;21(12):3148-59
references.pmid: 12065427
references.citation: EMBO J. 2002 Dec 16;21(24):6944-53
references.pmid: 12486015
references.citation: J Mol Biol. 2003 Jun 6;329(3):441-65
references.pmid: 12767828
references.citation: Mol Cell. 2003 Dec;12(6):1525-36
references.pmid: 14690605
references.citation: J Mol Biol. 1975 Sep 5;97(1):99-112
references.pmid: 1100854
references.citation: J Biol Chem. 1982 Nov 25;257(22):13770-5
references.pmid: 6292205
references.citation: Mol Gen Genet. 1987 Sep;209(2):382-90
references.pmid: 2959842
references.citation: J Mol Biol. 2008 Aug 29;381(2):249-55
references.pmid: 18602646
references.citation: PLoS One. 2008;3(8):e2984
references.pmid: 18714349
references.citation: Dig Dis Sci. 2009 Feb;54(2):282-91
references.pmid: 18465232
references.citation: Mol Cell. 2009 Nov 25;36(4):654-66
references.pmid: 19941825
references.citation: J Biol Chem. 2010 Jan 8;285(2):979-87
references.pmid: 19880515
references.citation: EMBO J. 2010 Jan 6;29(1):145-57
references.pmid: 19851282
references.citation: Mol Cell. 2010 Jan 29;37(2):247-58
references.pmid: 20122406
references.citation: Nucleic Acids Res. 2011 Feb;39(3):949-57
references.pmid: 20923786
references.citation: Nucleic Acids Res. 2011 Mar;39(4):1351-9
references.pmid: 20959294
references.citation: Cell. 2011 Aug 19;146(4):533-43
references.pmid: 21854980
references.citation: Science. 2011 Oct 14;334(6053):235-8
references.pmid: 21998391
references.citation: Cell. 2012 Oct 12;151(2):267-77
references.pmid: 23022319
references.citation: Adv Exp Med Biol. 2013;767:97-121
references.pmid: 23161008
references.citation: Cell. 2013 Apr 11;153(2):438-48
references.pmid: 23562643
references.citation: Proc Natl Acad Sci U S A. 2013 Apr 30;110(18):7252-7
references.pmid: 23589869
references.citation: Cold Spring Harb Perspect Biol. 2013 May;5(5):a012815
references.pmid: 23637285
references.citation: J Cell Sci. 2013 Mar 15;126(Pt 6):1297-306
references.pmid: 23645160
references.citation: PLoS One. 2013;8(7):e69607
references.pmid: 23874974
references.citation: Mol Microbiol. 1999 Oct;34(1):91-101
references.pmid: 10540288
references.citation: J Biol Chem. 1996 Jun 28;271(26):15656-61
references.pmid: 8663106
references.citation: J Biol Chem. 1996 Aug 30;271(35):21391-7
references.pmid: 8702920
references.citation: EMBO J. 1997 Jan 15;16(2):430-8
references.pmid: 9029161
references.citation: J Mol Biol. 1997 Jul 11;270(2):212-21
references.pmid: 9236123
references.citation: J Biol Chem. 1998 Jan 23;273(4):2452-7
references.pmid: 9442096
references.citation: Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3552-5
references.pmid: 10097074
references.citation: J Mol Biol. 1999 Jun 11;289(3):503-16
references.pmid: 10356325
references.citation: J Bacteriol. 1999 Aug;181(15):4549-53
references.pmid: 10419952
references.citation: J Biol Chem. 1999 Aug 27;274(35):25033-41
references.pmid: 10455182
references.citation: J Mol Biol. 2014 Dec 12;426(24):3917-28
references.pmid: 25308339
references.citation: Histopathology. 2015 Jan;66(2):192-200
references.pmid: 24805087
references.citation: Nucleic Acids Res. 2016 Jan 29;44(2):657-68
references.pmid: 26490956
references.citation: BMC Microbiol. 2004;4:47
references.pmid: 15588282
references.citation: Mol Cell. 2005 Mar 4;17(5):733-43
references.pmid: 15749022
references.citation: Mol Cell. 2005 Jul 22;19(2):247-58
references.pmid: 16039593
references.citation: J Biol Chem. 2005 Oct 7;280(40):34143-51
references.pmid: 16079128
references.citation: J Bacteriol. 1991 Nov;173(21):6686-93
references.pmid: 1938875
references.citation: J Biol Chem. 1991 Nov 25;266(33):22096-101
references.pmid: 1657989
references.citation: J Biol Chem. 1992 Apr 5;267(10):6933-40
references.pmid: 1313026
references.citation: J Bacteriol. 1995 Jul;177(14):4121-30
references.pmid: 7608087
references.citation: Mol Microbiol. 1995 Sep;17(5):825-31
references.pmid: 8596432
references.citation: Methods Enzymol. 1995;262:507-21
references.pmid: 8594376
references.citation: Methods Enzymol. 1995;262:604-13
references.pmid: 8594381
references.citation: Cell. 1996 Feb 23;84(4):643-50
references.pmid: 8598050
references.citation: Oncogene. 2006 Feb 16;25(7):1090-8
references.pmid: 16247466