Determination of protein-DNA binding constants and specificities from statistical analyses of single molecules: MutS-DNA interactions.
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.
Yang
Yong
Y
Department of Chemistry, University of North Carolina at Chapel Hill Chapel Hill, NC 27599-3290, USA.
Sass
Lauryn E
LE
Du
Chunwei
C
Hsieh
Peggy
P
Erie
Dorothy A
DA
D000251:Adenosine Triphosphatases / Q000378:metabolism* / Q000648:ultrastructure; D001426:Bacterial Proteins / Q000378:metabolism* / Q000648:ultrastructure; D020137:Base Pair Mismatch; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000648:ultrastructure; D003627:Data Interpretation, Statistical; D018625:Microscopy, Atomic Force; D051719:MutS DNA Mismatch-Binding Protein; D011485:Protein Binding
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D004247:DNA; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein