Title: Visualizing the One-Dimensional Diffusion of DNA Mismatch Repair Proteins at the Single-Molecule Level
Description: The eukaryotic post-replicative DNA mismatch repair pathway corrects mispaired bases that escape polymerase-proofreading machinery during DNA synthesis before the errors become permanently embedded in the genome. The initial steps in this essential pathway involve a series of specific target searches along DNA by the protein complexes Msh2-Msh6 and Mlh1-Pms1 in order to locate and remove mispaired bases. The details of these critical processes remain poorly understood, largely due to a lack of experimental methods capable of probing these dynamic processes. A custom total internal reflection fluorescence microscopy assay was developed to investigate these events by visualizing the proteins as they search along DNA in real time at the single molecule level. Both Msh2-Msh6 and Mlh1-Pms1 were found to travel along DNA by means of a one-dimensional random walk driven by thermal energy; however, the results presented in this work also reveal distinct mechanisms of diffusion utilized by each complex.

The observed diffusive behavior of Msh2-Msh6 is consistent with a model in which the protein forms a highly processive clamp that rotates about the DNA helical axis as it diffuses, thereby remaining in register with the phosphate backbone as it scans DNA for mispaired bases. Mlh1-Pms1 movements are more consistent with a loosely bound ring-like structure that moves along DNA by a hopping or stepping mechanism as it searches for mismatch-bound Msh2-Msh6. These assays provide critical novel insights into the initial steps of mismatch recognition, address long-debated models proposed for post-recognition events in mismatch repair and also provide a platform for studying the protein-protein interactions of Msh2-Msh6 with Mlh1-Pms1 following mismatch recognition. In addition to providing important details into the DNA repair pathway itself, the data also reveal distinct limitations different modes of diffusion may impose on DNA target searches in vivo and these results can be generalized to various other DNA-binding proteins that move along DNA by similar mechanisms.

Importantly, this work also provides the first experimental evidence directly observing unbiased facilitated diffusion as a mechanism of target search and recognition by any protein. The manner by which DNA-binding proteins are able to survey a vast amount of nonspecific genomic DNA in order to recognize a small number of specific sites or structures is a fundamental issue in understanding a diverse array of protein-DNA processes such as replication, gene expression and DNA repair and the diversity of targets and necessary search mechanisms involved in the mismatch repair pathway make it an excellent model system for studying facilitated diffusion along DNA.