Asymmetric ATP binding and hydrolysis activity of the Thermus aquaticus MutS dimer is key to modulation of its interactions with mismatched DNA.
Prokaryotic MutS and eukaryotic Msh proteins recognize base pair mismatches and insertions or deletions in DNA and initiate mismatch repair. These proteins function as dimers (and perhaps higher order oligomers) and possess an ATPase activity that is essential for DNA repair. Previous studies of Escherichia coli MutS and eukaryotic Msh2-Msh6 proteins have revealed asymmetry within the dimer with respect to both DNA binding and ATPase activities. We have found the Thermus aquaticus MutS protein amenable to detailed investigation of the nature and role of this asymmetry. Here, we show that (a) in a MutS dimer one subunit (S1) binds nucleotide with high affinity and the other (S2) with 10-fold weaker affinity, (b) S1 hydrolyzes ATP rapidly while S2 hydrolyzes ATP at a 30-50-fold slower rate, (c) mismatched DNA binding to MutS inhibits ATP hydrolysis at S1 but slow hydrolysis continues at S2, and (d) interaction between mismatched DNA and MutS is weakened when both subunits are occupied by ATP but remains stable when S1 is occupied by ATP and S2 by ADP. These results reveal key MutS species in the ATPase pathway; S1(ADP)-S2(ATP) is formed preferentially in the absence of DNA or in the presence of fully matched DNA, while S1(ATP)-S2(ATP) and S1(ATP)-S2(ADP) are formed preferentially in the presence of mismatched DNA. These MutS species exhibit differences in interaction with mismatched DNA that are likely important for the mechanism of MutS action in DNA repair.
Antony
Edwin
E
Molecular Biology and Biochemistry Department, Wesleyan University, 205 Hall-Atwater Laboratories, Middletown, Connecticut 06459, USA.
Hingorani
Manju M
MM
D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D020137:Base Pair Mismatch / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D004794:Enzyme Repression / Q000235:genetics; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D007700:Kinetics; D051719:MutS DNA Mismatch-Binding Protein; D016254:Mutagenesis, Insertional; D011485:Protein Binding / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D013824:Thermus / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D021122:Protein Subunits; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli