title: Multiple C-terminal tails within a single E. coli SSB homotetramer coordinate DNA replication and repair.
abstract: Escherichia coli single-stranded DNA binding protein (SSB) plays essential roles in DNA replication, recombination and repair. SSB functions as a homotetramer with each subunit possessing a DNA binding domain (OB-fold) and an intrinsically disordered C-terminus, of which the last nine amino acids provide the site for interaction with at least a dozen other proteins that function in DNA metabolism. To examine how many C-termini are needed for SSB function, we engineered covalently linked forms of SSB that possess only one or two C-termini within a four-OB-fold "tetramer". Whereas E. coli expressing SSB with only two tails can survive, expression of a single-tailed SSB is dominant lethal. E. coli expressing only the two-tailed SSB recovers faster from exposure to DNA damaging agents but accumulates more mutations. A single-tailed SSB shows defects in coupled leading and lagging strand DNA replication and does not support replication restart in vitro. These deficiencies in vitro provide a plausible explanation for the lethality observed in vivo. These results indicate that a single SSB tetramer must interact simultaneously with multiple protein partners during some essential roles in genome maintenance.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, Box 8231, St. Louis, MO 63110-1093, USA.
authors.forename: Edwin
authors.initials: E
authors.lastname: Antony
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Weiland
authors.forename: Quan
authors.initials: Q
authors.lastname: Yuan
authors.forename: Carol M
authors.initials: CM
authors.lastname: Manhart
authors.forename: Binh
authors.initials: B
authors.lastname: Nguyen
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Kozlov
authors.forename: Charles S
authors.initials: CS
authors.lastname: McHenry
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Lohman
mesh_terms: D004252:DNA Mutational Analysis; D004260:DNA Repair; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D050296:Microbial Viability; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C476430:SSB protein, E coli
keywords: DNA binding; DNA repair; DNA replication; EDTA; FRET; PBS; SIP; SSB; SSB interacting protein; ethylenediaminetetraacetic acid; fluorescence resonance energy transfer; phosphate-buffered saline; single stranded DNA binding protein; single-stranded DNA; single-stranded DNA binding protein; ssDNA; wild type; wt
references.citation: Biochemistry. 2010 Sep 28;49(38):8266-75
references.pmid: 20795631
references.citation: J Biol Chem. 1982 May 25;257(10):5692-9
references.pmid: 7040370
references.citation: Nature. 2009 Oct 22;461(7267):1092-7
references.pmid: 19820696
references.citation: Nucleic Acids Res. 2006 Feb 18;34(4):1084-91
references.pmid: 16488881
references.citation: EMBO J. 1998 Apr 15;17(8):2436-49
references.pmid: 9545254
references.citation: Anal Biochem. 2004 Mar 15;326(2):234-56
references.pmid: 15003564
references.citation: Annu Rev Biochem. 1994;63:527-70
references.pmid: 7979247
references.citation: Cell. 2011 Jul 22;146(2):222-32
references.pmid: 21784244
references.citation: Cell. 1996 Feb 23;84(4):643-50
references.pmid: 8598050
references.citation: J Biol Chem. 1996 Aug 30;271(35):21398-405
references.pmid: 8702921
references.citation: J Biol Chem. 2005 Dec 2;280(48):39693-700
references.pmid: 16188886
references.citation: J Biol Chem. 2009 Nov 13;284(46):31672-9
references.pmid: 19749191
references.citation: Annu Rev Biochem. 2011;80:403-36
references.pmid: 21675919
references.citation: J Mol Biol. 1986 Feb 20;187(4):603-15
references.pmid: 3519979
references.citation: Nature. 1950 Dec 30;166(4235):1112-4
references.pmid: 14796714
references.citation: J Mol Biol. 1989 May 5;207(1):249-68
references.pmid: 2661832
references.citation: J Bacteriol. 1975 Feb;121(2):511-7
references.pmid: 1089633
references.citation: Biochemistry. 1986 Dec 2;25(24):7799-802
references.pmid: 3542037
references.citation: Nat Struct Biol. 2000 Aug;7(8):648-52
references.pmid: 10932248
references.citation: J Biol Chem. 1996 Jun 28;271(26):15656-61
references.pmid: 8663106
references.citation: J Mol Biol. 1991 Jan 5;217(1):63-74
references.pmid: 1988680
references.citation: Trends Biochem Sci. 1988 Jul;13(7):250-5
references.pmid: 2855682
references.citation: J Biol Chem. 1992 Feb 25;267(6):4054-63
references.pmid: 1531480
references.citation: J Biol Chem. 2008 Mar 28;283(13):8274-82
references.pmid: 18223256
references.citation: Nucleic Acids Res. 2004 Dec 02;32(21):6378-87
references.pmid: 15576682
references.citation: Nucleic Acids Res. 2011 Aug;39(15):6536-45
references.pmid: 21572106
references.citation: EMBO J. 2007 Oct 3;26(19):4239-51
references.pmid: 17853894
references.citation: J Biol Chem. 1994 Nov 25;269(47):30005-13
references.pmid: 7962001
references.citation: J Mol Biol. 1994 Feb 11;236(1):106-23
references.pmid: 8107097
references.citation: J Biol Chem. 1989 Jul 5;264(19):11294-301
references.pmid: 2567734
references.citation: Proc Natl Acad Sci U S A. 1982 Oct;79(19):5803-7
references.pmid: 6764531
references.citation: Cold Spring Harb Symp Quant Biol. 1984;49:553-9
references.pmid: 6397310
references.citation: J Gen Appl Microbiol. 1997 Dec;43(6):309-316
references.pmid: 12501301
references.citation: J Mol Biol. 1989 May 5;207(1):269-88
references.pmid: 2661833
references.citation: Biochemistry. 2002 May 14;41(19):6032-44
references.pmid: 11993998
references.citation: Nucleic Acids Res. 2012 Jul;40(13):6039-48
references.pmid: 22447448
references.citation: Biophys J. 1998 Sep;75(3):1503-12
references.pmid: 9726952
references.citation: Nat Rev Genet. 2001 Jul;2(7):504-15
references.pmid: 11433357
references.citation: J Biol Chem. 1985 Mar 25;260(6):3594-603
references.pmid: 3882711
references.citation: Biochem Pharmacol. 1988 May 1;37(9):1781-2
references.pmid: 3288214
references.citation: J Mol Biol. 1981 Jan 5;145(1):75-104
references.pmid: 7265204
references.citation: Radioisotopes. 1984 May;33(5):307-14
references.pmid: 6382455
references.citation: PLoS Genet. 2010 Dec 09;6(12):e1001238
references.pmid: 21170359
references.citation: J Biol Chem. 1988 Apr 5;263(10):4629-40
references.pmid: 3280566
references.citation: Methods Enzymol. 2004;384:185-212
references.pmid: 15081688
references.citation: Trends Biochem Sci. 2000 Apr;25(4):185-9
references.pmid: 10754552
references.citation: Biochemistry. 2002 Oct 1;41(39):11611-27
references.pmid: 12269804
references.citation: Mol Cell. 2010 Feb 26;37(4):481-91
references.pmid: 20188667
references.citation: Microbiol Rev. 1994 Sep;58(3):401-65
references.pmid: 7968921
references.citation: J Biol Chem. 2007 Jun 29;282(26):19247-58
references.pmid: 17483090
references.citation: Mol Cell. 2007 Jun 22;26(6):781-93
references.pmid: 17588514
references.citation: J Biol Chem. 2000 May 19;275(20):15512-9
references.pmid: 10748120
references.citation: CRC Crit Rev Biochem. 1986;19(3):191-245
references.pmid: 3512164
references.citation: J Biol Chem. 1998 Sep 4;273(36):23476-84
references.pmid: 9722585
references.citation: Nucleic Acids Res. 2005 Mar 21;33(5):1662-70
references.pmid: 15781492
references.citation: Nucleic Acids Res. 2006;34(22):6708-17
references.pmid: 17148487
references.citation: Biochim Biophys Acta. 1966 Mar 21;114(3):501-15
references.pmid: 5330658
references.citation: Methods Enzymol. 1995;262:507-21
references.pmid: 8594376
references.citation: Cell. 1999 Jan 8;96(1):153-63
references.pmid: 9989506
references.citation: Nature. 1968 Oct 19;220(5164):276-7
references.pmid: 4879326
references.citation: J Biol Chem. 1996 Aug 30;271(35):21391-7
references.pmid: 8702920
references.citation: Nucleic Acids Res. 1996 Jul 15;24(14):2706-11
references.pmid: 8759000
references.citation: J Biol Chem. 2009 Jan 30;284(5):3264-3272
references.pmid: 18986990
references.citation: J Biol Chem. 1986 Sep 5;261(25):11460-5
references.pmid: 3528143
references.citation: Methods Mol Biol. 2012;922:133-49
references.pmid: 22976181
references.citation: J Biol Chem. 2013 Feb 8;288(6):3989-99
references.pmid: 23264623
references.citation: Microbiol Rev. 1990 Dec;54(4):342-80
references.pmid: 2087220
references.citation: Mol Cell. 2010 Jan 29;37(2):273-81
references.pmid: 20122408
references.citation: J Biol Chem. 2012 Oct 12;287(42):35621-35630
references.pmid: 22902627
references.citation: J Biol Chem. 2001 Feb 9;276(6):4441-6
references.pmid: 11078744
references.citation: J Mol Biol. 2007 Jun 22;369(5):1244-57
references.pmid: 17490681
references.citation: Biochim Biophys Acta. 1965 Jan 11;95:181-3
references.pmid: 14289027
references.citation: Nucleic Acids Res. 2013 Apr;41(8):4507-17
references.pmid: 23430154
references.citation: Biochemistry. 1988 Apr 5;27(7):2260-5
references.pmid: 3289611
references.citation: Crit Rev Biochem Mol Biol. 2008 Sep-Oct;43(5):289-318
references.pmid: 18937104
references.citation: J Biol Chem. 1992 Feb 25;267(6):4045-53
references.pmid: 1740452
references.citation: J Biol Chem. 2001 May 18;276(20):16992-7
references.pmid: 11279060
references.citation: J Mol Biol. 2009 Feb 27;386(3):612-25
references.pmid: 19150358
references.citation: EMBO J. 2011 Aug 19;30(20):4236-47
references.pmid: 21857649
references.citation: Annu Rev Biochem. 1986;55:103-36
references.pmid: 3527040
references.citation: Biotechnology (N Y). 1990 Jan;8(1):47-51
references.pmid: 1366512
references.citation: J Biol Chem. 1983 Mar 10;258(5):3346-55
references.pmid: 6298232
references.citation: Mol Microbiol. 1997 Jul;25(2):237-46
references.pmid: 9282736
references.citation: Proc Natl Acad Sci U S A. 1974 Oct;71(10):3858-62
references.pmid: 4610564
references.citation: J Bacteriol. 1991 Apr;173(8):2720-3
references.pmid: 2013585
references.citation: Proc Natl Acad Sci U S A. 1997 Jun 24;94(13):6652-7
references.pmid: 9192620
references.citation: Proc Natl Acad Sci U S A. 2004 Jun 8;101(23):8575-80
references.pmid: 15159541
references.citation: J Biol Chem. 2012 Jun 22;287(26):22123-32
references.pmid: 22570477
references.citation: Biochemistry. 2010 May 4;49(17):3555-66
references.pmid: 20329707