DNA Chain Length Dependence of Formation and Dynamics of hMutSα·hMutLα·Heteroduplex Complexes

• Published in: The Journal of biological chemistry 2001-08, Vol.276 (35), p.33233-33240
• Main Authors: Blackwell, Leonard J., Wang, Shuntai, Modrich, Paul
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M105076200

Formation of a ternary complex between human MutSα, MutLα, and heteroduplex DNA has been demonstrated by surface plasmon resonance spectroscopy and electrophoretic gel shift methods. Formation of the hMutLα·hMutSα·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 ∼0.8 mol of hMutLα/mol of heteroduplex-bound hMutSα. Although the steady-state levels of the hMutLα·hMutSα· 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 hMutSα, the hMutLα·hMutSα complex can migrate along the helix contour to dissociate at DNA ends.