Trapping DNA-protein binding reactions with neutral osmolytes for the analysis by gel mobility shift and self-cleavage assays.
We take advantage of our previous observation that neutral osmolytes can strongly slow down the rate of DNA-protein complex dissociation to develop a method that uses osmotic stress to 'freeze' mixtures of DNA-protein complexes and prevent further reaction enabling analysis of the products. We apply this approach to the gel mobility shift assay and use it to modify a self-cleavage assay that uses the nuclease activity of the restriction endonucleases to measure sensitively their specific binding to DNA. At sufficiently high concentrations of neutral osmolytes the cleavage reaction can be triggered at only those DNA fragments with initially bound enzyme. The self-cleavage assay allows measurement of binding equilibrium and kinetics directly in solution avoiding the intrinsic problems of gel mobility shift and filter binding assays while providing the same sensitivity level. Here we compare the self-cleavage and gel mobility shift assays applied to the DNA binding of EcoRI and BamHI restriction endonucleases. Initial results indicate that BamHI dissociation from its specific DNA sequence is strongly linked to water activity with the half-life time of the specific complex increasing approximately 20-fold from 0 to 1 osmolal betaine.
Sidorova
Nina Y
NY
Laboratory of Physical and Structural Biology, NICHD, National Institutes of Health, Bethesda, MD 20892, USA. sidorova@mail.nih.gov
Muradymov
Shakir
S
Rau
Donald C
DC
D001622:Betaine / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D015245:Deoxyribonuclease BamHI / Q000032:analysis* / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000032:analysis* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D007700:Kinetics; D009997:Osmotic Pressure; D011485:Protein Binding
D004268:DNA-Binding Proteins; D001622:Betaine; D004247:DNA; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI