Trapping DNA-protein binding reactions with neutral osmolytes for the analysis by gel mobility shift and self-cleavage assays

Abstract

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 ∼20-fold from 0 to 1 osmolal betaine.

Differences in Hydration Coupled to Specific and Nonspecific Competitive Binding and to Specific DNA Binding of the Restriction Endonuclease BamHI

“…Our goal here is 2-fold: 1) to measure directly differences in sequestered water between specific and nonspecific BamHI complexes to confirm the correspondence between waters measured by osmotic stress and structure; and 2) to demonstrate the complications connected with using osmotic stress for reactions that are accompanied by significant changes in exposed surface area as, for example, the specific binding of free protein. We measure the ratio of specific and nonspecific binding constants, K nsp-sp , 2 by direct competition assayed with a novel self-cleavage assay developed by us (16). The osmotic pressure dependence of K nsp-sp indicates that about 120 -150 extra water molecules are retained in the nonspecific versus specific BamHI-DNA complex in very good agreement with the x-ray structural data.…”

“…We use an approach termed the osmotic stress technique (2, 3) that measures changes in hydration coupled with changes in the functional state by measuring the effect of water activity on reaction equilibria and kinetics. The osmotic stress technique has been used previously to measure the changes in hydration accompanying the DNA binding of several regulatory proteins: Escherichia coli gal (4), lac (5), tyr (6), and Cro (7) repressors, E. coli CAP protein (8), Hin recombinase (9), Ultrathorax and Deformed homeodomains (10), the restriction endonucleases , BamHI (15,16), and EcoRV (17), HhaI methyltransferase (18), Sso7d protein (19), the TATA-binding protein (20,21), and the E2C protein from papillomavirus (22).The dependence of an equilibrium constant on the bulk solution osmotic pressure that is varied by adding neutral solutes that do not bind directly to the DNA or protein gives a difference in the number of associated waters between the initial reactants and the final products. More precisely, water is considered associated with the DNA, protein, and complex if it excludes osmolyte, otherwise there is no osmotic imbalance.…”

“…We use an approach termed the osmotic stress technique (2,3) that measures changes in hydration coupled with changes in the functional state by measuring the effect of water activity on reaction equilibria and kinetics. The osmotic stress technique has been used previously to measure the changes in hydration accompanying the DNA binding of several regulatory proteins: Escherichia coli gal (4), lac (5), tyr (6), and Cro (7) repressors, E. coli CAP protein (8), Hin recombinase (9), Ultrathorax and Deformed homeodomains (10), the restriction endonucleases EcoRI (11)(12)(13)(14), BamHI (15,16), and EcoRV (17), HhaI methyltransferase (18), Sso7d protein (19), the TATA-binding protein (20,21), and the E2C protein from papillomavirus (22).…”

A SILAC-based DNA protein interaction screen that identifies candidate binding proteins to functional DNA elements

“…In comparison to the EMSA technique, our solid phase approach is much less subjected to nonphysiological buffer conditions (ionic strength and pH), which occur during gel electrophoresis and can drastically disturb the equilibrium between free DNA and protein-DNA complexes (Sidorova et al 2005). Thus, interactions revealed by our SILAC-based screen in vitro will most likely also have the potential to occur in vivo, where they are of course influenced by additional levels of regulation (chromatin structure and histone modifications).…”

Biological Wonders of Osmolytes: The Need to Know More

“…The effect was observed to be concentration and/or solvent condition dependent. Potential of osmolytes in trapping DNA-protein binding reactions with natural osmolytes has been reported [81]. The reason for this trapping has been assigned to slowing down of the rate of dissociation of the complex of the nucleic acid with the protein.…”