Single-molecule imaging reveals target-search mechanisms during DNA mismatch repair

The ability of proteins to locate specific targets among a vast excess of nonspecific DNA is a fundamental theme in biology. Basic principles governing these search mechanisms remain poorly understood, and no study has provided direct visualization of single proteins searching for and engaging target sites. Here we use the postreplicative mismatch repair proteins MutS alpha and MutL alpha as model systems for understanding diffusion-based target searches. Using single-molecule microscopy, we directly visualize MutS alpha as it searches for DNA lesions, MutL alpha as it searches for lesion-bound MutS alpha, and the MutS alpha/MutL alpha complex as it scans the flanking DNA. We also show that MutL alpha undergoes intersite transfer between juxtaposed DNA segments while searching for lesion-bound MutS alpha, but this activity is suppressed upon association with MutS alpha, ensuring that MutS/MutL remains associated with the damage-bearing strand while scanning the flanking DNA. Our findings highlight a hierarchy of lesion- and ATP-dependent transitions involving both MutS alpha and MutL alpha, and help establish how different modes of diffusion can be used during recognition and repair of damaged DNA.