Determination of protein–DNA binding constants and specificities from statistical analyses of single molecules: MutS–DNA interactions
| Autor: | Chunwei Du, Peggy Hsieh, Dorothy A. Erie, Yong Yang, Lauryn E. Sass |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2005 |
| Předmět: |
MutS DNA Mismatch-Binding Protein
Base Pair Mismatch Plasma protein binding Biology Microscopy Atomic Force DNA-binding protein Article 03 medical and health sciences chemistry.chemical_compound Bacterial Proteins Genetics Binding site Binding selectivity 030304 developmental biology Adenosine Triphosphatases 0303 health sciences Binding Sites 030302 biochemistry & molecular biology DNA Affinities DNA-Binding Proteins Biochemistry chemistry Data Interpretation Statistical Biophysics Protein Binding |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | Atomic force microscopy (AFM) is a powerful technique for examining the conformations of protein-DNA complexes and determining the stoichiometries and affinities of protein-protein complexes. We extend the capabilities of AFM to the determination of protein-DNA binding constants and specificities. The distribution of positions of the protein on the DNA fragments provides a direct measure of specificity and requires no knowledge of the absolute binding constants. The fractional occupancies of the protein at a given position in conjunction with the protein and DNA concentrations permit the determination of the absolute binding constants. We present the theoretical basis for this analysis and demonstrate its utility by characterizing the interaction of MutS with DNA fragments containing either no mismatch or a single mismatch. We show that MutS has significantly higher specificities for mismatches than was previously suggested from bulk studies and that the apparent low specificities are the result of high affinity binding to DNA ends. These results resolve the puzzle of the apparent low binding specificity of MutS with the expected high repair specificities. In conclusion, from a single set of AFM experiments, it is possible to determine the binding affinity, specificity and stoichiometry, as well as the conformational properties of the protein-DNA complexes. |
| Databáze: | OpenAIRE |
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