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Papers In Press, published online ahead of print July 5, 2001
Biochemistry and HHMI, Duke Univ. Medical Center, Durham, NC 27710
Corresponding Author: modrich{at}biochem.duke.edu
Formation of a ternary complex between human MutSa, MutLa, and heteroduplex DNA has been demonstrated by surface plasmon resonance spectroscopy and electrophoretic gel shift methods. Formation of the hMutLa•hMutSa•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 less effective, and a 41 base pair heteroduplex was inactive. As judged by surface plasmon resonance spectroscopy, ternary complexes produced with the 200 base pair G-T DNA contained approximately 0.8 mol hMutLa per mol of heteroduplex-bound hMutSa. Although the steady-state levels of the hMutLa•hMutSa•heteroduplex were substantial, this complex was found to turn over as judged by surface plasmon resonance spectroscopy and electrophoretic gel shift analysis. With the former method, the majority of the complexes dissociated rapidly upon termination of protein flow, and dissociation occurred in the latter case upon challenge with competitor DNA. However, ternary complex dissociation as monitored by gel shift assay was prevented if both ends of the heteroduplex were physically blocked with streptavidin•biotin complexes. This observation suggests that like hMutSa, the hMutLa•hMutSa complex can migrate along the helix contour to dissociate at DNA ends.
J. Biol. Chem, 10.1074/jbc.M105076200
Submitted on June 3, 2001
Revised on July 3, 2001
Accepted on July 3, 2001
DNA chain length dependence of formation and dynamics of hMutSa5hMutLa5 heteroduplex complexes
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