Rapid kinetic measurements of MutS‐mismatched DNA interactions

MutS, an essential and highly conserved dimeric DNA repair protein, binds mismatches and insertion/deletion loops in DNA to initiate the process of mismatch repair. The MutS subunits S 1 and S 2 exhibit asymmetric ATP binding and hydrolysis activities that appear linked to their asymmetric mismatch binding activity. We are investigating the kinetics of MutS‐DNA interactions in order to understand this link in greater detail. Our DNA substrates contain 2‐aminopurine adjacent to the mismatched base pair; its fluorescence reports interaction with the MutS protein. We find that MutS binds to a mismatched DNA rapidly (3 x 10 6 M −1 s −1 ), while no interaction can be detected with fully matched DNA. Ongoing experiments examine MutS binding to (and dissociation from) mismatched DNA substrates when the S 1 and/or S 2 sites are free or bound by ADP, ATP, and the non‐hydrolysable ATP analogue ATPγS. Preliminary results indicate that the presence and identity of the two occupying nucleotides modulate the mismatch binding activity (and likely the repair activity) of MutS. Funding: Barry M. Goldwater Scholarship and Excellence in Education Program, Howard Hughes Medical Institute, and National Institutes of Health.