Homology recognition without double-stranded DNA-str and separ ation in D-loop formation by RecA

Abstract

RecA protein and RecA / Rad51 orthologues are required for homologous recombination and DNA repair in all living creatures. RecA / Rad51 catalyz es f ormation of the D-loop, an obligatory recombination intermediate, through an ATP-dependent reaction consisting of two phases: homology recognition between double-stranded (ds)DNA and single-stranded (ss)DNA to form a h ybrid-duple x core of 6-8 base pairs and sub- sequent h ybrid-duple x / D-loop processing. Ho w dsDNA recogniz es homologous ssDNA is contro v ersial. T he aromatic residue at the tip of the β-hairpin loop (L2) was shown to stabilize dsDNA-strand separation. We tested a model in which dsDNA strands were separated by the aromatic residue before homology recognition and found that the aromatic residue was not essential to homology recognition, but was required for D-loop processing. Contrary to the model, we found that the double helix was not unwound even a single turn during search for sequence homology, but rather w as un w ound only after the homologous sequence was recognized. These results suggest that dsDNA recognizes its homologous ssDNA before strand separation. The search for homologous sequence with homologous ssDNA without dsDNA-strand separation does not generate stress within the dsDNA; this would be an advantage for dsDNA to express homology-dependent functions in vivo and also in vitro.