Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 276, Issue 35, Pages 33233-33240Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M105076200
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- NIGMS NIH HHS [GM45190] Funding Source: Medline
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Formation of a ternary complex between human MutS alpha, MutL alpha, and heteroduplex DNA has been demonstrated by surface plasmon resonance spectroscopy 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 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 similar to0.8 mol of hMutL alpha /mol of heteroduplex-bound hMutS alpha. Although the steady-state levels of the hMutL alpha (.)hMutS alpha (.)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 hMutL alpha (.)hMutS alpha complex can migrate along the helix contour to dissociate at DNA ends.
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