RecA as a motor protein. Testing models for the role of ATP hydrolysis in DNA strand exchange.

Abstract

ATP hydrolysis (by RecA protein) fundamentally alters the properties of RecA protein-mediated DNA strand exchange reactions. ATP hydrolysis renders DNA strand exchange unidirectional, greatly increases the lengths of hybrid DNA created, permits the bypass of heterologous DNA insertions in one or both DNA substrates, and is absolutely required for exchange reactions involving four DNA strands. There are at least two viable models to explain how ATP hydrolysis is coupled to DNA strand exchange so as to bring about these effects. The first couples ATP hydrolysis to a redistribution of RecA monomers within a RecA filament. The second couples ATP hydrolysis to a facilitated rotation of the DNA substrates. The RecA monomer redistribution model makes the prediction that heterology bypass should not occur if the single-stranded DNA substrate is linear. The facilitated DNA rotation model predicts that RecA protein should promote the separation of paired DNA strands within a RecA filament if one of them is contiguous with a length of DNA being rotated about the filament exterior. Here, a facile bypass of heterologous insertions with linear DNA substrates is demonstrated, providing evidence against a role for RecA monomer redistribution in heterology bypass. In addition, we demonstrate that following a four-strand DNA exchange reaction, a distal segment of DNA hundreds of base pairs in length can be unwound in a nonreciprocal phase of the reaction, consistent with the direct coupling of an ATP hydrolytic motor to the proposed DNA rotation.