Figure 3.

The number of MSH sliding clamps affects the rate of LacI-binding association. The binding kinetics of LacI (1 nm) to LacO was directly measured by smPIFE as described previously (31) and correlated with the number of TaMutS (315 nm) and EcMutS (40 nm) sliding clamps stably bound to the mismatched DNA after washing unbound MutS. a, an illustration of the mismatched DNA substrate. The 98-bp DNA containing a 15T+ mismatch was similar to Fig. 2 except the DNA contained a Cy3 fluorophore located 11 nt from the biotin–streptavidin capable of PIFE upon LacI binding, and the TaMutS and EcMutS were labeled with Alexa Fluor 647 as described (supporting information). b, representative trace showing PIFE in the Cy3 channel upon LacI binding (τ_off indicated) and the number of Alexa Fluor 647 fluorophore photobleaching events associated with the same DNA for one fluorophore (top) and two fluorophores (bottom). c, the k_on·LacI for LacI only, for LacI in the presence of one or two TaMutS sliding clamps, and for LacI in the presence of one or two EcMutS sliding clamps (± S.D.). The k_on·LacI was determined from the τ_off·LacI data shown in supporting Table S3 and color-coded similarly as follows: red hash, least squares calculation using one-fluorophore data; blue hash, least squares calculation using two-fluorophore data; green hash, maximum likelihood calculation using one-fluorophore data; purple hash, maximum likelihood calculation using two-fluorophore data (supporting information).