Variations of intramolecular ligation rates allow the detection of protein-induced bends in DNA.

Abstract

A method requiring minute amounts of DNA and protein is proposed for the detection of DNA bending in solution. Local bending, induced by the binding of a protein at its stereospecific site, must increase the probability of circularization of short DNA fragments and hence the rate of formation of the corresponding minicircles. This effect should be highly sensitive to the location of the protein‐binding site on the DNA fragment. Simple controls allow other possible interpretations to be ruled out. The deformation due to the cyclic AMP receptor protein when it interacts with its stereospecific site at the lactose control region is studied by this method. It is shown in this case that untwisting is negligible and bending significant. Further measurements are required to assess the actual value of the radius of curvature of the DNA double helix in this complex.

The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures.

“…The HMG proteins could be promoting invertasome assembly by binding to specific DNA structures, by directly stabilizing protein complexes, or by facilitating looping of the DNA between protein-binding sites. To investigate the latter possibility, we employed a ligation assay that has been used by others to demonstrate DNA-bending activity (Kotlarz et al 1986;Dripps and Wartell 1987;Zahn and Blattner 1987;Hodges-Garcia et al 1989;Schroth et al 1989). In this assay, bending is measured by the circularization of a DNA substrate shorter than the average persistence length (-150 bp), which will not circularize in the absence of DNA-bending protein.…”

Mammalian SMC3 C-terminal and Coiled-coil Protein Domains Specifically Bind Palindromic DNA, Do Not Block DNA Ends, and Prevent DNA Bending

“…The assay was performed essentially as described (27)(28)(29)(30)(31)(32)(33)(34). Reaction mixtures (10 l) contained 20 mM HEPES (pH 7.5), 1 mM DTT, 100 g/ml bovine serum albumin, 0.5-1 nM 189-bp fragment, and protein as indicated.…”

“…For investigating DNA bending by proteins, including those that do not bind a specific DNA sequence, the ring closure or circularization assay has been widely used (27)(28)(29)(30)(31)(32)(33)(34). This assay, whose theoretical foundation was worked out in 1981 (27), generally uses an end-labeled, linear DNA substrate of a length between 70 and 200 bp with cohesive ends.…”

“…Due to the limited length and the resulting steric tensions, such fragments are not easily circularized to allow intramolecular ligation. DNA fragments shorter than about 150 bp behave as stiff molecules in solution and can hardly be ligated intramolecularly (28,36). Proteins that bend DNA, however, can efficiently support intramolecular ligation, i.e.…”

Protein-induced bending and DNA cyclization.

“…Rates or product distributions of ligase-catalyzed cyclization are affected by the binding of CAP (25,26), HU (27), and cro repressor (28). In this paper, we show quantitatively that Reactions were initiated by the addition of 7.5 ,ul of diluted ligase for a final volume of 75 ,ul, and eight 8-pl time points over 15-30 min were quenched by addition to 4 pl of 75 mM EDTA, proteinase K at 2 mg/ml, 15% glycerol, and dyes.…”