Gel-Shift Assays: Migrative Dissociation of a RecA−Oligonucleotide Complex during Electrophoresis in Hydroxyethylated Agarose Gels

Abstract

We investigate the dynamics of a protein-DNA complex under gel-shift assay conditions, in particular how the degree of gel confinement influences the complex stability during the electrophoretic analysis. The fiber-like complex between RecA proteins and a single-stranded oligonucleotide has been studied in hydroxyethylated agarose gels with the average pore radius being between 1 and 5 times larger than the radius of the rodshaped complex. The confining effect of the gel matrix slows down the dissociation of the complex, but in addition, migrative interactions (collisions) with the gel network during the electrophoresis perturbs the complex in two ways. At low gel concentrations, the protein-DNA complex is dismantled into free RecA and oligonucleotide with a half-time of the complex that decreases with increasing field strength in accordance with a migrative mechanism. At a given field strength, the half-time increases with increasing gel concentration despite more frequent interactions, probably because of a counteracting stabilizing cage effect from the gel. At high gel concentrations, a second type of perturbation is reflected in an increasing complex velocity over time, probably caused by trains of end-to-end attached fibers being broken up by interactions with the tight gels.