RecA-promoted sliding of base pairs within DNA repeats: quantitative analysis by a slippage assay.
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.
Navadgi
Vasundhara M
VM
Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India.
Sen
Subhojit
S
Rao
Basuthkar J
BJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D009841:Oligonucleotides / Q000494:pharmacology; D011485:Protein Binding; D011938:Rec A Recombinases / Q000378:metabolism*; D013997:Time Factors
D009841:Oligonucleotides; D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases