Further evidence for binding of three single-stranded DNA molecules by the RecA filament.

Abstract

The ability to accommodate three single-strands of DNA by each filamentous RecA-DNA complex, in the presence of the non-hydrolysable ATP analog, adenosine 5'-O-3-thiotriphosphate (ATP gamma S), was demonstrated by monitoring the fast renaturation that occurs between the complementary homopolymers, poly(dA) and poly(dT), upon sodium dodecyl sulfate (SDS)-induced dissociation of RecA. The renaturation could be followed from the linear dichroism signal of DNA. The reaction was fast and complete when SDS was added to a mixture of RecA having three nucleobases per subunit each of poly(dA) and poly(dT), while the reaction was slower and incomplete in the absence of RecA. The efficient renaturation indicates that the two DNA strands were bound close to each other, i.e., to the same RecA filament. Similar fast renaturation was observed upon the addition of SDS to a mixture of RecA with three bases each of poly(dA), poly(dT), and a non-complementary DNA, poly(dC), even when one of the complementary DNAs was added last as the third strand. Since the dissociation of DNA from RecA is extremely slow in the presence of ATP-gamma S, and thus no renaturation can occur within the RecA filaments, the results indicate that RecA can bind three single-stranded DNA molecules.