RecA-promoted sliding of base pairs within DNA repeats: quantitative analysis by a slippage assay

Abstract

RecA that catalyses efficient homology search and exchange of DNA bases has to effect major transitions in the structure as well as the dynamics of bases within RecA–DNA filament. RecA induces slippage of paired strands in poly(dA)–poly(dT) duplex using the energy of ATP hydrolysis. Here, we have adopted the targeted ligation assay and quantified the strand slippage within a short central cassette of (dA)₄–(dT)₄ duplex. The design offers a stringent test case for scoring a cross-talk between A residues with those of T that are diagonally placed on the opposite strand at either −3, −2, −1, +1, +2, or +3 pairing frames. As expected, the cross-talk levels in RecA mediated as well as thermally annealed duplexes were maximal in non-diagonal pairing frame (i.e., 0-frame), the levels of which fell off gradually as the frames became more diagonal, i.e., −3<−2<−1 or +3<+2<+1. Interestingly, the level of cross-talk in naked duplexes was intrinsically less efficient in minus frames than their plus frame counterparts. The asymmetry created in naked duplexes by such a disparity between minus versus plus frames was partially obviated by RecA. Moreover, RecA promoted a significantly higher level of cross-talk selectively in −2 and −1 frames, as compared to that in naked DNA, which suggests a model that the elevated cross-talk in RecA filament may be limited to base pairs housed within the same rather than adjacent RecA monomers.