The effects of nucleotides on MutS-DNA binding kinetics clarify the role of MutS ATPase activity in mismatch repair.
MutS protein initiates mismatch repair with recognition of a non-Watson-Crick base-pair or base insertion/deletion site in DNA, and its interactions with DNA are modulated by ATPase activity. Here, we present a kinetic analysis of these interactions, including the effects of ATP binding and hydrolysis, reported directly from the mismatch site by 2-aminopurine fluorescence. When free of nucleotides, the Thermus aquaticus MutS dimer binds a mismatch rapidly (k(ON)=3 x 10(6) M(-1) s(-1)) and forms a stable complex with a half-life of 10 s (k(OFF)=0.07 s(-1)). When one or both nucleotide-binding sites on the MutS*mismatch complex are occupied by ATP, the complex remains fairly stable, with a half-life of 5-7 s (k(OFF)=0.1-0.14 s(-1)), although MutS(ATP) becomes incapable of (re-)binding the mismatch. When one or both nucleotide-binding sites on the MutS dimer are occupied by ADP, the MutS*mismatch complex forms rapidly (k(ON)=7.3 x 10(6) M(-1) s(-1)) and also dissociates rapidly, with a half-life of 0.4 s (k(OFF)=1.7 s(-1)). Integration of these MutS DNA-binding kinetics with previously described ATPase kinetics reveals that: (a) in the absence of a mismatch, MutS in the ADP-bound form engages in highly dynamic interactions with DNA, perhaps probing base-pairs for errors; (b) in the presence of a mismatch, MutS stabilized in the ATP-bound form releases the mismatch slowly, perhaps allowing for onsite interactions with downstream repair proteins; (c) ATP-bound MutS then moves off the mismatch, perhaps as a mobile clamp facilitating repair reactions at distant sites on DNA, until ATP is hydrolyzed (or dissociates) and the protein turns over.
Jacobs-Palmer
Emily
E
Wesleyan University, Molecular Biology and Biochemistry Department, Middletown, CT 06459, USA.
Hingorani
Manju M
MM
D015075:2-Aminopurine / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D020137:Base Pair Mismatch; D001483:Base Sequence; D053843:DNA Mismatch Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D008957:Models, Genetic; D008969:Molecular Sequence Data; D015394:Molecular Structure; D051719:MutS DNA Mismatch-Binding Protein / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry
D004268:DNA-Binding Proteins; D009711:Nucleotides; D015075:2-Aminopurine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein