Detection of High-Affinity and Sliding Clamp Modes for MSH2-MSH6 by Single-Molecule Unzipping Force Analysis

Abstract

Mismatch repair (MMR) is initiated by MutS family proteins (MSH) that recognize DNA mismatches and recruit downstream repair factors. We used a single-molecule DNA-unzipping assay to probe interactions between S. cerevisiae MSH2-MSH6 and a variety of DNA mismatch substrates. This work revealed a high-specificity binding state of MSH proteins for mismatch DNA that was not observed in bulk assays and allowed us to measure the affinity of MSH2-MSH6 for mismatch DNA as well as its footprint on DNA surrounding the mismatch site. Unzipping analysis with mismatch substrates containing an end blocked by lac repressor allowed us to identify MSH proteins present on DNA between the mismatch and the block, presumably in an ATP-dependent sliding clamp mode. These studies provide a high-resolution approach to study MSH interactions with DNA mismatches and supply evidence to support and refute different models proposed for initiation steps in MMR.

DNA Looping Mediates Nucleosome Transfer

“…DNA tethers were formed in flow chambers and were unzipped using an optical trap with a loading rate clamp of 10 pN s À 1 ( Fig. 1 and Supplementary Figs 3 and 5) through the bound protein [10][11][12]15,16,[28][29][30][31]55,[57][58][59][60] . Briefly, chambers were first incubated with antidigoxygenin at 0.2 mg ml À 1 for 5 min, and then the surface was blocked by incubation with casein at 5 mg ml À 1 for 5 min.…”

Human MSH2 (hMSH2) Protein Controls ATP Processing by hMSH2-hMSH6

“…ADP 3 ATP exchange is rate-limiting and provokes a conformational transition that results in the formation of a sliding clamp, which dissociates from the mismatch (8,11). One possible model for MMR suggests that mismatch-dependent loading of multiple hydrolysis-independent sliding clamps activates downstream MMR components (8,(12)(13)(14), whereas another model suggests hydrolysis-dependent translocation to contact downstream MMR components (15)(16)(17). The management of ATP processing by the two MSH subunits is predicted to be significantly different for these two models.…”

Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and looping

“…The unwinding of the DNA is inherent to the function of helicases such as RNA polymerase (9), the mismatch repair MutS proteins (10) and HSV-1 UL9 protein (11,12), and hence these helicases must track the DNA. However, in the case of restriction enzymes (13) and the chromatin remodelling Snf2 proteins (14), there is no functional requirement to unwind the DNA.…”