Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 366, Issue 4, Pages 1087-1098Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2006.11.092
Keywords
mismatch repair; MutS; DNA binding; ATPase; kinetic mechanism
Categories
Funding
- NIGMS NIH HHS [R01 GM064514-01, GM64514-01, R01 GM064514] Funding Source: Medline
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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 center dot 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 center dot 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 n-dsmatch 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. (c) 2006 Elsevier Ltd. All rights reserved.
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