title: Roles of type 1A topoisomerases in genome maintenance in Escherichia coli.
abstract: In eukaryotes, type 1A topoisomerases (topos) act with RecQ-like helicases to maintain the stability of the genome. Despite having been the first type 1A enzymes to be discovered, much less is known about the involvement of the E. coli topo I (topA) and III (topB) enzymes in genome maintenance. These enzymes are thought to have distinct cellular functions: topo I regulates supercoiling and R-loop formation, and topo III is involved in chromosome segregation. To better characterize their roles in genome maintenance, we have used genetic approaches including suppressor screens, combined with microscopy for the examination of cell morphology and nucleoid shape. We show that topA mutants can suffer from growth-inhibitory and supercoiling-dependent chromosome segregation defects. These problems are corrected by deleting recA or recQ but not by deleting recJ or recO, indicating that the RecF pathway is not involved. Rather, our data suggest that RecQ acts with a type 1A topo on RecA-generated recombination intermediates because: 1-topo III overproduction corrects the defects and 2-recQ deletion and topo IIII overproduction are epistatic to recA deletion. The segregation defects are also linked to over-replication, as they are significantly alleviated by an oriC::aph suppressor mutation which is oriC-competent in topA null but not in isogenic topA+ cells. When both topo I and topo III are missing, excess supercoiling triggers growth inhibition that correlates with the formation of extremely long filaments fully packed with unsegregated and diffuse DNA. These phenotypes are likely related to replication from R-loops as they are corrected by overproducing RNase HI or by genetic suppressors of double topA rnhA mutants affecting constitutive stable DNA replication, dnaT::aph and rne::aph, which initiates from R-loops. Thus, bacterial type 1A topos maintain the stability of the genome (i) by preventing over-replication originating from oriC (topo I alone) and R-loops and (ii) by acting with RecQ.
authors.affiliation: Département de microbiologie, infectiologie et immunologie, Université de Montréal, Succ. Centre-ville, Montréal, Québec, Canada.
authors.forename: Valentine
authors.initials: V
authors.lastname: Usongo
authors.affiliation: Département de microbiologie, infectiologie et immunologie, Université de Montréal, Succ. Centre-ville, Montréal, Québec, Canada.
authors.forename: Marc
authors.initials: M
authors.lastname: Drolet
mesh_terms: D004265:DNA Helicases / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D016680:Genome, Bacterial / Q000235:genetics; D042822:Genomic Instability; D053484:RecQ Helicases / Q000235:genetics*
chemical_list: C506724:RecQ protein, E coli; D004265:DNA Helicases; D053484:RecQ Helicases; D004264:DNA Topoisomerases, Type I
references.citation: J Bacteriol. 2008 Nov;190(22):7346-56
references.pmid: 18790862
references.citation: J Bacteriol. 1999 May;181(10):3185-92
references.pmid: 10322021
references.citation: Front Biosci. 2003 Jan 01;8:d256-63
references.pmid: 12456368
references.citation: Cell. 1998 Oct 30;95(3):419-30
references.pmid: 9814711
references.citation: Mol Cell. 1999 May;3(5):611-20
references.pmid: 10360177
references.citation: Nature. 2011 Oct 02;478(7368):209-13
references.pmid: 21964332
references.citation: Mol Microbiol. 2012 Nov;86(4):1007-22
references.pmid: 23066834
references.citation: DNA Repair (Amst). 2010 Mar 2;9(3):202-9
references.pmid: 20097140
references.citation: J Biol Chem. 1997 Jan 17;272(3):1473-9
references.pmid: 8999816
references.citation: FEMS Microbiol Lett. 2003 Apr 25;221(2):237-42
references.pmid: 12725933
references.citation: Mol Cell. 2007 Apr 27;26(2):273-86
references.pmid: 17466628
references.citation: J Mol Biol. 2001 Mar 2;306(4):669-79
references.pmid: 11243779
references.citation: Microbiol Mol Biol Rev. 1997 Jun;61(2):212-38
references.pmid: 9184011
references.citation: J Biol Chem. 1994 Jan 21;269(3):2068-74
references.pmid: 8294458
references.citation: J Biol Chem. 1999 Jun 4;274(23):16659-64
references.pmid: 10347234
references.citation: EMBO J. 1999 Mar 15;18(6):1701-11
references.pmid: 10075939
references.citation: J Biol Chem. 1986 Apr 25;261(12):5616-24
references.pmid: 3514619
references.citation: Mol Microbiol. 2006 Feb;59(3):723-30
references.pmid: 16420346
references.citation: Mol Microbiol. 2011 Dec;82(6):1330-48
references.pmid: 22026368
references.citation: Nat Rev Cancer. 2009 Sep;9(9):644-54
references.pmid: 19657341
references.citation: Genetics. 2005 Apr;169(4):1799-806
references.pmid: 15716497
references.citation: Mol Microbiol. 2005 Oct;58(1):80-101
references.pmid: 16164551
references.citation: Mol Gen Genet. 1984;195(1-2):170-4
references.pmid: 6092846
references.citation: Nucleic Acids Res. 2012 Feb;40(4):1648-65
references.pmid: 22053082
references.citation: Cell. 1989 Jul 28;58(2):409-19
references.pmid: 2546682
references.citation: Mol Gen Genet. 1978 Jun 1;162(1):9-16
references.pmid: 353526
references.citation: J Biol Chem. 2000 Apr 14;275(15):11257-63
references.pmid: 10753935
references.citation: J Bacteriol. 1978 Dec;136(3):1008-17
references.pmid: 363684
references.citation: Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3526-30
references.pmid: 7536935
references.citation: Annu Rev Biochem. 2013;82:139-70
references.pmid: 23495937
references.citation: J Mol Biol. 1999 Nov 26;294(2):321-32
references.pmid: 10610761
references.citation: J Mol Biol. 1985 Oct 5;185(3):625-37
references.pmid: 2997454
references.citation: Mol Microbiol. 2000 Jan;35(1):58-68
references.pmid: 10632877
references.citation: J Biol Chem. 1997 May 9;272(19):12816-23
references.pmid: 9139742
references.citation: Annu Rev Biochem. 2001;70:369-413
references.pmid: 11395412
references.citation: EMBO J. 1995 May 15;14(10):2385-92
references.pmid: 7774596
references.citation: Mol Microbiol. 2008 Dec;70(6):1323-33
references.pmid: 19019156
references.citation: Cell. 1982 Nov;31(1):35-42
references.pmid: 6297751
references.citation: Mol Cell. 2014 Feb 6;53(3):484-97
references.pmid: 24507716
references.citation: Mol Biosyst. 2008 Nov;4(11):1075-84
references.pmid: 18931783
references.citation: J Biol Chem. 2003 Aug 15;278(33):30705-10
references.pmid: 12788950
references.citation: Nature. 2013 Aug 29;500(7464):608-11
references.pmid: 23892781
references.citation: Proc Natl Acad Sci U S A. 1976 Nov;73(11):3872-6
references.pmid: 186775
references.citation: J Bacteriol. 1994 Sep;176(17):5494-504
references.pmid: 8071228
references.citation: Annu Rev Microbiol. 2011;65:19-35
references.pmid: 21639790
references.citation: Mol Microbiol. 2007 Sep;65(6):1485-92
references.pmid: 17824928
references.citation: PLoS Genet. 2012;8(4):e1002622
references.pmid: 22496668
references.citation: Mol Microbiol. 2003 May;48(3):845-54
references.pmid: 12694626
references.citation: Genes Dev. 2010 Jul 15;24(14):1546-58
references.pmid: 20634320
references.citation: Cell. 1982 Nov;31(1):43-51
references.pmid: 6297752
references.citation: J Mol Biol. 1971 Feb 14;55(3):523-33
references.pmid: 4927945
references.citation: J Bacteriol. 2008 Sep;190(17):5995-6001
references.pmid: 18567657
references.citation: J Biol Chem. 2003 Oct 24;278(43):42668-78
references.pmid: 12909639
references.citation: Genes Dev. 2000 Feb 1;14(3):360-5
references.pmid: 10673507
references.citation: Nature. 2003 Dec 18;426(6968):870-4
references.pmid: 14685245
references.citation: Mol Microbiol. 2007 Apr;64(1):125-38
references.pmid: 17376077
references.citation: J Biol Chem. 2000 Mar 17;275(11):8103-13
references.pmid: 10713132
references.citation: J Biol Chem. 2012 Jun 1;287(23):19346-53
references.pmid: 22511792
references.citation: Mol Microbiol. 2004 Oct;54(1):198-211
references.pmid: 15458416
references.citation: Mol Microbiol. 2011 Dec;82(6):1305-10
references.pmid: 22074454
references.citation: J Biol Chem. 1988 Sep 15;263(26):13366-73
references.pmid: 2843517
references.citation: J Biol Chem. 1984 Jul 10;259(13):8578-83
references.pmid: 6330114
references.citation: Mol Cell Biol. 1994 Dec;14(12):8391-8
references.pmid: 7969174
references.citation: EMBO J. 2005 Jul 20;24(14):2679-87
references.pmid: 15990871
references.citation: Mol Gen Genet. 1980 Jan;177(2):301-9
references.pmid: 6245341
references.citation: J Biol Chem. 2000 Mar 31;275(13):9636-44
references.pmid: 10734115
references.citation: Proc Natl Acad Sci U S A. 2001 Aug 14;98(17):9766-71
references.pmid: 11493711
references.citation: J Biol Chem. 1989 Oct 25;264(30):17924-30
references.pmid: 2553698
references.citation: Mol Microbiol. 2008 Aug;69(4):968-81
references.pmid: 18554330
references.citation: Proc Natl Acad Sci U S A. 2006 Jul 25;103(30):11118-23
references.pmid: 16849422
references.citation: Proc Natl Acad Sci U S A. 1987 Oct;84(20):7024-7
references.pmid: 2823250
references.citation: Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4068-73
references.pmid: 16537486
references.citation: Mol Microbiol. 2009 Oct;74(2):467-79
references.pmid: 19737351
references.citation: Biochimie. 2007 Apr;89(4):427-46
references.pmid: 17293019
references.citation: Genetics. 2003 Apr;163(4):1255-71
references.pmid: 12702673
references.citation: J Bacteriol. 2013 Apr;195(8):1758-68
references.pmid: 23396913
references.citation: EMBO J. 2011 Aug 19;30(20):4236-47
references.pmid: 21857649
references.citation: Proc Natl Acad Sci U S A. 1984 Feb;81(4):1040-4
references.pmid: 6322184
references.citation: Mol Cell Proteomics. 2012 Aug;11(8):492-500
references.pmid: 22535208
references.citation: EMBO J. 1997 Jan 15;16(2):430-8
references.pmid: 9029161
references.citation: Nat Struct Mol Biol. 2010 Nov;17(11):1377-82
references.pmid: 20935631
references.citation: Arch Microbiol. 2002 Sep;178(3):193-201
references.pmid: 12189420
references.citation: Mol Microbiol. 2000 Feb;35(4):888-95
references.pmid: 10692165
references.citation: Mol Cell. 2008 Jun 20;30(6):779-89
references.pmid: 18570879
references.citation: J Antimicrob Chemother. 2010 May;65(5):924-30
references.pmid: 20228083
references.citation: Genetics. 2002 Oct;162(2):647-62
references.pmid: 12399378
references.citation: J Biol Chem. 1999 Jun 4;274(23):16654-8
references.pmid: 10347233
references.citation: Genes Dev. 2000 Mar 15;14(6):740-9
references.pmid: 10733533