BPS2026 – Single-molecule tracking shows orchestration of the DNA mismatch repair pathway in live bacteria

The range of suitable fluorophores for live-cell single-molecule tracking has grown substantially in recent years. Improvements in the photostability of fluorescent HaloTag ligands provide the exciting opportunity to track an individual protein over unprecedented timescales. Such advances are particularly advantageous in the study of DNA repair, where single-molecule tracking can be used to directly observe DNA repair proteins searching for and dwelling at sites of DNA damage. We exploit the distinctive photophysical properties of two synthetic dyes, namely tetramethylrhodamine (TMR) and JFX650, to obtain complementary insights into DNA repair mechanisms. With its stochastic photoswitching cycles, TMR generates thousands of short (∼1s) observations per cell, enabling an overview of the diffusive dynamics for the whole protein population. In contrast, the supreme photostability of JFX650 permits tracking a single molecule per cell over ∼minute timescales, thereby resolving complete DNA lesion search and repair cycles. We apply both approaches to investigate the molecular mechanisms underpinning the DNA mismatch repair (MMR) pathway in E. coli . By combining single-molecule tracking with strategic genetic perturbations and cellular treatments, we obtain mechanistic insights into how MutS, MutL, and MutH enzymes are recruited to DNA mismatches and how their interactions orchestrate the repair pathway. Dual-color imaging further allows us to localize MMR in relation to the DNA replication fork. Our results suggest an updated model for MMR in E. coli.