Single-trajectory Bayesian modeling reveals multi-state diffusion of the MSH sliding clamp

DNA mismatch repair (MMR) is an essential mechanism for preserving genomic integrity across diverse living organisms. In this process, MutS homologs (MSH) play crucial roles in detecting mismatched basepairs and recruiting downstream MMR proteins. MSH exhibits distinct functions and diffusion dynamics before and after mismatch recognition. The ADP-bound MSH, known as the searching clamp, scans DNA via rotational diffusion along the backbone, while ATP binding produces a stable sliding clamp. Recent experiments have challenged the conventional view that the ATP-bound clamp performs a simple Brownian motion. Here, we investigate the diffusion dynamics of the ATP-bound MSH sliding clamp using single-particle tracking and a Bayesian diffusion-state analysis framework. Our quantitative modeling reveals that the diffusion characteristics defy explanation by a single-state diffusion mechanism. Instead, we identify three discrete diffusion states with distinct coefficients ( D 1 = 1.86 × 10 −2 μm 2 /s, D 2 = 1.30 × 10 −1 μm 2 /s, and D 3 = 9.64 × 10 −1 μm 2 /s) and cross-state transitions predominantly mediated via the intermediate D 2 -state. We propose that these multi-state dynamics reflect conformational switching in the MSH clamp, highlighting a more intricate and regulated diffusion mechanism than previously recognized.