Banaz, N. | bioRxiv Science

bioRxiv2026

Replication-coupled search positions MutH for strand incision in DNA mismatch repair

DNA mismatch repair (MMR) safeguards genome stability by correcting replication errors, yet how its components coordinate as an efficient pathway in living cells remains unclear. In Escherichia coli, MutS and MutL detect mismatches and must rapidly activate MutH to incise the nascent DNA strand at distantly located GATC sites. Here, we used live-cell single-molecule tracking to resolve the target-search dynamics of MMR proteins in vivo. We find that MutH binds DNA at hemimethylated GATC sites independently of mismatch recognition and without MutS or MutL. The autonomous recruitment of MutH is facilitated by a switch from fast diffusion to a slow-search mode near DNA replication forks. This guidance allows MutH to transiently occupy potential incision sites in the wake of replication forks, awaiting activation by MutS-MutL. The parallelised search mechanism of MMR proteins facilitates rapid and targeted repair of replication errors in cells.

molecular biology↗

bioRxiv2018

Choosing the right label for single-molecule tracking in live bacteria: Side-by-side comparison of photoactivatable fluorescent protein and Halo tag dyes

Visualizing and quantifying molecular motion and interactions inside living cells provides crucial insight into the mechanisms underlying cell function. This has been achieved by super-resolution localization microscopy and single-molecule tracking in conjunction with photoactivatable fluorescent proteins. An alternative labelling approach relies on genetically-encoded protein tags with cell-permeable fluorescent ligands which are brighter and less prone to photobleaching than fluorescent proteins but require a laborious labelling process. Either labelling method is associated with significant advantages and disadvantages that should be taken into consideration depending on the microscopy experiment planned. Here, we describe an optimised procedure for labelling Halo-tagged proteins in live Escherichia coli cells. We provide a side-by-side comparison of Halo tag with different fluorescent ligands against the popular photoactivatable fluorescent protein PAmCherry. Using test proteins with different intracellular dynamics, we evaluated fluorescence intensity, background, photostability, and single-molecule localization and tracking results. Capitalising on the brightness and extended spectral range of fluorescent Halo ligands, we also demonstrate high-speed and dual-colour single-molecule tracking.

biophysics↗