The role of nucleotide cofactor binding in cooperativity and specificity of MutS recognition.
Mismatch repair (MMR) is essential for eliminating biosynthetic errors generated during replication or genetic recombination in virtually all organisms. The critical first step in Escherichia coli MMR is the specific recognition and binding of MutS to a heteroduplex, containing either a mismatch or an insertion/deletion loop of up to four nucleotides. All known MutS homologs recognize a similar broad spectrum of substrates. Binding and hydrolysis of nucleotide cofactors by the MutS-heteroduplex complex are required for downstream MMR activity, although the exact role of the nucleotide cofactors is less clear. Here, we showed that MutS bound to a 30-bp heteroduplex containing an unpaired T with a binding affinity approximately 400-fold stronger than to a 30-bp homoduplex, a much higher specificity than previously reported. The binding of nucleotide cofactors decreased both MutS specific and nonspecific binding affinity, with the latter marked by a larger drop, further increasing MutS specificity by approximately 3-fold. Kinetic studies showed that the difference in MutS K(d) for various heteroduplexes was attributable to the difference in intrinsic dissociation rate of a particular MutS-heteroduplex complex. Furthermore, the kinetic association event of MutS binding to heteroduplexes was marked by positive cooperativity. Our studies showed that the positive cooperativity in MutS binding was modulated by the binding of nucleotide cofactors. The binding of nucleotide cofactors transformed E. coli MutS tetramers, the functional unit in E. coli MMR, from a cooperative to a noncooperative binding form. Finally, we found that E. coli MutS bound to single-strand DNA with significant affinity, which could have important implication for strand discrimination in eukaryotic MMR mechanism.
Huang
Shar-yin N
SN
Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, P.O. Box 208114, New Haven, CT 06520-8114, USA.
Crothers
Donald M
DM
D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D001483:Base Sequence; D001667:Binding, Competitive / Q000187:drug effects; D003067:Coenzymes / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009692:Nucleic Acid Heteroduplexes; D009711:Nucleotides / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D017186:Titrimetry
D003067:Coenzymes; D050505:Mutant Proteins; D009692:Nucleic Acid Heteroduplexes; D009711:Nucleotides; D000266:Adenylyl Imidodiphosphate; D051719:MutS DNA Mismatch-Binding Protein
J Biol Chem. 1998 Sep 4;273(36):23176-82
9722547
Biotechniques. 1995 Dec;19(6):956-65
8747662
Prog Nucleic Acid Res Mol Biol. 2001;66:159-202
11051764
J Biol Chem. 2004 Jul 2;279(27):28402-10
15105434
Annu Rev Biochem. 2005;74:681-710
15952900
Gastroenterology. 1993 May;104(5):1535-49
8482467
EMBO J. 1997 Jul 16;16(14):4467-76
9250691
J Biol Chem. 2004 Oct 15;279(42):43879-85
15297450
Annu Rev Microbiol. 2003;57:579-608
14527292
Curr Opin Genet Dev. 1997 Feb;7(1):105-13
9024626
Nature. 1989 Nov 23;342(6248):396-401
2555716
Immunity. 1998 Jul;9(1):135-41
9697843
Science. 1992 May 8;256(5058):784-9
1589758
Proc Natl Acad Sci U S A. 2003 Dec 9;100(25):14822-7
14634210
J Biol Chem. 2003 Dec 5;278(49):49505-11
14506224
Mol Cell. 2001 Jan;7(1):1-12
11172706
Annu Rev Genet. 1976;10:135-56
797306
Biochemistry. 2000 Mar 21;39(11):3176-83
10715140
Nature. 2000 Oct 12;407(6805):711-7
11048711
Mol Cell Biol. 1989 Oct;9(10):4432-40
2685551
Biochem J. 2001 Mar 15;354(Pt 3):627-33
11237867
Biochemistry. 2004 Oct 19;43(41):13115-28
15476405
Proc Natl Acad Sci U S A. 1986 Jul;83(14):5057-61
3014530
Hum Mutat. 1996;8(1):1-7
8807329
Nature. 2000 Oct 12;407(6805):703-10
11048710
Mol Cell. 2007 May 25;26(4):579-92
17531815
J Mol Biol. 2007 Mar 2;366(4):1087-98
17207499
J Biol Chem. 2001 Sep 7;276(36):34339-47
11454861
Annu Rev Biochem. 1996;65:101-33
8811176
J Biol Chem. 2003 Sep 5;278(36):34667-73
12829697
Mol Cell Biol. 1997 May;17(5):2436-47
9111312
J Biol Chem. 2003 May 16;278(20):18557-62
12624105
Hum Mutat. 2003 Jun;21(6):577-81
12754702
Mol Cell. 1999 Feb;3(2):255-61
10078208
J Bacteriol. 2005 Sep;187(18):6577-9
16159793
Nucleic Acids Res. 1997 Jun 15;25(12):2245-54
9171073
Nucleic Acids Res. 2005 Aug 01;33(13):4322-34
16061937
J Biol Chem. 1999 Aug 13;274(33):23673-8
10438551
J Natl Cancer Inst. 1996 Sep 18;88(18):1317-9
8797773
J Clin Oncol. 2003 Mar 15;21(6):1174-9
12637487
Nature. 1993 Apr 15;362(6421):652-4
8464518
J Biol Chem. 2001 Jul 27;276(30):28291-9
11371566
Carcinogenesis. 1997 May;18(5):889-96
9163672
Nucleic Acids Res. 2003 Aug 15;31(16):4814-21
12907723
J Biol Chem. 2001 Dec 7;276(49):45505-8
11602569
Nucleic Acids Res. 1998 Sep 15;26(18):4291-300
9722651
J Mol Biol. 2001 Jan 26;305(4):805-16
11162093
J Biol Chem. 1988 May 15;263(14):6829-35
2834393
J Mol Biol. 2003 Dec 12;334(5):949-65
14643659
J Biol Chem. 2007 Jun 1;282(22):16345-54
17426027