UmuD(2) inhibits a non-covalent step during DinB-mediated template slippage on homopolymeric nucleotide runs.
Escherichia coli DinB (DNA polymerase IV) possesses an enzyme architecture resulting in specialized lesion bypass function and the potential for creating -1 frameshifts in homopolymeric nucleotide runs. We have previously shown that the mutagenic potential of DinB is regulated by the DNA damage response protein UmuD(2). In the current study, we employ a pre-steady-state fluorescence approach to gain a mechanistic understanding of DinB regulation by UmuD(2). Our results suggest that DinB, like its mammalian and archaeal orthologs, uses a template slippage mechanism to create single base deletions on homopolymeric runs. With 2-aminopurine as a fluorescent reporter in the DNA substrate, the template slippage reaction results in a prechemistry fluorescence change that is inhibited by UmuD(2). We propose a model in which DNA templates containing homopolymeric nucleotide runs, when bound to DinB, are in an equilibrium between non-slipped and slipped conformations. UmuD(2), when bound to DinB, displaces the equilibrium in favor of the non-slipped conformation, thereby preventing frameshifting and potentially enhancing DinB activity on non-slipped substrates.
Foti
James J
JJ
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Delucia
Angela M
AM
Joyce
Catherine M
CM
Walker
Graham C
GC
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011938:Rec A Recombinases / Q000378:metabolism; D012697:Serine Endopeptidases / Q000494:pharmacology*; D013698:Templates, Genetic
C094399:DinB protein, E coli; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D009711:Nucleotides; D004247:DNA; D011938:Rec A Recombinases; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase; C055218:UmuD protein, E coli; D012697:Serine Endopeptidases
Mol Cell. 2007 Dec 28;28(6):1058-70
18158902
J Biol Chem. 2002 Aug 2;277(31):28157-66
12023283
Proc Natl Acad Sci U S A. 2007 Sep 18;104(38):14905-10
17848527
Genes Cells. 1999 Nov;4(11):607-18
10620008
Nature. 2006 Jan 12;439(7073):225-8
16407906
Genes Dev. 2000 Jul 1;14(13):1589-94
10887153
Mol Gen Genet. 1991 Sep;229(1):10-6
1654503
Mol Cell. 2004 Mar 12;13(5):751-62
15023344
Biochemistry. 2007 Sep 25;46(38):10790-803
17725324
J Biol Chem. 2006 Jan 27;281(4):2358-72
16306039
Genes Dev. 2003 Sep 1;17(17):2191-9
12952891
PLoS Biol. 2006 Jan;4(1):e11
16379496
Proc Natl Acad Sci U S A. 2008 Jan 29;105(4):1152-7
18216271
Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13792-7
9391106
EMBO Rep. 2008 May;9(5):459-64
18369368
J Biol Chem. 2002 Sep 13;277(37):34198-207
12097328
Mol Cell. 2007 Feb 23;25(4):601-14
17317631
Cold Spring Harb Symp Quant Biol. 1966;31:77-84
5237214
Nucleic Acids Res. 2008 Nov;36(20):6571-84
18931375
J Biol Chem. 2002 Feb 15;277(7):5265-74
11734560
Mol Cell. 2008 Mar 28;29(6):767-79
18374650
Genetics. 1990 Jun;125(2):275-80
2199309
J Biol Chem. 2004 Oct 29;279(44):45360-8
15339923
J Biol Chem. 1997 Jan 24;272(4):2559-69
8999973
Nucleic Acids Res. 2000 Nov 1;28(21):4138-46
11058110
Proc Natl Acad Sci U S A. 2009 Dec 15;106(50):21137-42
19948952
J Biol Chem. 2000 Dec 15;275(50):39678-84
11006276
Proc Natl Acad Sci U S A. 1990 Jul;87(13):4946-50
2195542
J Biol Chem. 1988 Oct 15;263(29):14784-9
3049589
Genetics. 1991 Aug;128(4):695-701
1916241
Mol Cell Biol. 2004 Jul;24(13):5687-93
15199127
J Biol Chem. 2004 Jul 30;279(31):32932-40
15155753
J Biol Chem. 1997 Oct 31;272(44):27919-30
9346941
Mol Cell. 2001 Jul;8(1):7-8
11515498
J Biol Chem. 2009 Aug 14;284(33):22467-22480
19542228
J Biol Chem. 2002 May 31;277(22):19633-8
11919199
Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8342-9
11459973
Nucleic Acids Res. 2000 Nov 1;28(21):4147-56
11058111
J Biol Chem. 1986 Oct 15;261(29):13581-7
3759982
Biochemistry. 2001 Jan 30;40(4):946-56
11170416