DNA chain length dependence of formation and dynamics of hMutSalpha.hMutLalpha.heteroduplex complexes.
Formation of a ternary complex between human MutSalpha, MutLalpha, and heteroduplex DNA has been demonstrated by surface plasmon resonance spectroscopy and electrophoretic gel shift methods. Formation of the hMutLalpha.hMutSalpha.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 of hMutLalpha/mol of heteroduplex-bound hMutSalpha. Although the steady-state levels of the hMutLalpha.hMutSalpha. 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 hMutSalpha, the hMutLalpha.hMutSalpha complex can migrate along the helix contour to dissociate at DNA ends.
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Related Subject Headings
- Thymine
- Surface Plasmon Resonance
- Proto-Oncogene Proteins
- Nucleic Acid Heteroduplexes
- Nuclear Proteins
- Neoplasm Proteins
- MutS Homolog 2 Protein
- MutL Protein Homolog 1
- Mismatch Repair Endonuclease PMS2
- Kinetics
Citation
Published In
DOI
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Thymine
- Surface Plasmon Resonance
- Proto-Oncogene Proteins
- Nucleic Acid Heteroduplexes
- Nuclear Proteins
- Neoplasm Proteins
- MutS Homolog 2 Protein
- MutL Protein Homolog 1
- Mismatch Repair Endonuclease PMS2
- Kinetics