Figure 4.
Concentration dependence of loaded TaMutS sliding clamps compared with theoretical Tonks Gas/Sliding Clamp model predictions. a, an illustration of the substrates utilized to count TaMutS sliding clamps on the DNA. A 95-bp duplex DNA was anchored to the smTIRF flow cell surface by a biotin-NeutrAvidin at one end while the other end was blocked by a dig-antidig complex. Cy5 was linked to the 7th base from the dig-antidig and the mismatch was located at the 15th bp position from the 5′-biotin end. b, representative traces showing the anti-correlation between Cy3–TaMutS and Cy5–DNA intensities caused by time-averaged FRET as well as photobleaching steps of Cy3–TaMutS emission. c, comparison between the fraction of TaMutS predicted by the Tonks Gas/Sliding Clamp model (solid line) and experimental number of TaMutS (circles ± S.E.) determined at three different TaMutS concentrations (10, 50, and 300 nm). d, the reduction in the ratio of LacI on-rate with zero MutS sliding clamps over the LacI on-rate with N number of MutS sliding clamps (kon·LacI-0/kon·LacI-N) with different DNA lengths. e, the mean (line) and upper and lower quartile (boxes) for kon·LacI-0/kon·LacI-N was determined for a 98-bp mismatched DNA (Fig. 3) using individual assumptions for the MSH footprint (σ = 24, 25, or 26 bp), LacI footprints (σ = 21 or 25 bp) or the possibility that either the 5′-biotin-NeutrAvidin or dig-antidig linkages increased the DNA length by an additional 2 bp. The red asterisks indicate the experimentally observed ratio from Fig. 3c.