DNA chain length dependence of formation and dynamics of hMutS alpha(.)hMutL alpha(.)heteroduplex complexes

Formation of a ternary complex between human MutS alpha, MutL alpha, and he teroduplex DNA has been demonstrated by surface plasmon resonance spectrosc opy and electrophoretic gel shift methods. Formation of the hMutL alpha (.) -hMutS alpha -heteroduplex complex requires a mismatch and ATP hydrolysis, and depends on DNA chain length. Ternary complex formation was supported by a 200-base pair G-T heteroduplex, a 100-base pair substrate was somewhat l ess effective, and a 41-base pair heteroduplex was inactive. As judged by s urface plasmon resonance spectroscopy, ternary complexes produced with the 200-base pair G-T DNA contained similar to0.8 mol of hMutL alpha /mol of he teroduplex-bound hMutS alpha. Although the steady-state levels of the hMutL alpha (.)hMutS alpha (.)heteroduplex were substantial, this complex was fo und to turn over, as judged by surface plasmon resonance spectroscopy and e lectrophoretic gel shift analysis. With the former method, the majority of the complexes dissociated rapidly upon termination of protein flow, and dis sociation occurred in the latter case upon challenge with competitor DNA. H owever, ternary complex dissociation as monitored by gel shift assay was pr evented if both ends of the heteroduplex were physically blocked with strep tavidin-biotin complexes. This observation suggests that, like hMutSa, the hMutL alpha (.)hMutS alpha complex can migrate along the helix contour to d issociate at DNA ends.