Modern Concepts in Evolutionary Genetics

Proceedings of the conference. ABSTRACT RecA protein catalyzes DNA strand exchange, a basic step of homologous recombination. RecA protein possesses two DNA binding sites. During DNA strand exchange, the primary site binds to ssDNA, forming the helical RecA nucleoprotein filament. The weaker secondary site binds dsDNA during the homology search process. Surprisingly, the secondary DNA binding site displays a higher affinity for ssDNA than for dsDNA. Binding of ssDNA to this secondary site shows base compositional specificity, in that polypyrimidinic DNA binds to the RecA protein-ssDNA filament with higher affinity than polypurinic sequences. We found that this specific-ity reflects an intrinsic property of the secondary site of RecA protein rather than an interaction between DNA molecules within nucleoprotein filament , i.e., self-recognition. Here we demonstrate that this site has a second important function. It binds the ssDNA strand that is displaced from ho-mologous duplex DNA during DNA strand exchange, stabilizing the initial heteroduplex DNA product. Although the high affinity of the secondary site for ssDNA is essential for DNA strand exchange , it renders DNA strand exchange sensitive to an excess of ssDNA which competes with dsDNA for binding. We further demonstrate that SSB can sequester ssDNA, preventing its binding to the secondary site and thereby assisting at two levels: it averts the inhibition caused by an excess of ssDNA and prevents the reversal of DNA strand exchange by removing the displaced strand from the secondary site.