Homologous recognition promoted by RecA protein via

The RecA protein of Escherichia coil forms a nucleoprotein filament that promotes homologous recognition and subsequent strand exchange between a single strand and duplex DNA via a three-stranded intermediate. Recognition of homology within three-stranded nucleoprotein complexes, which is probably central to genetic recombination, is not well understood as compared with the mutual recognition of complementary single strands by Watson-Crick base pairing. Using oligonucleotides, we examined the determinants of homologous recognition within RecA nucleoprotein filaments. Filaments that contained a single strand of DNA recognized homology not only in a complementary oligonucleotide but also in an identical oligonucleotide, whether their respective sugarphosphate backbones were antiparailel or parallel, and a filament that contained duplex DNA showed the same polymorphic versatility in the recognition ofhomology. Recognition of self by a filament that contains a single strand reveals that RecA fiaments can recognize homology via non-Watson-Crick hydrogen bonds. Recognition of multiple forms of the same sequence by duplex DNA in the fiament shows that it primarily senses base-sequence homology, and suggests that recognition can be accomplished prior to the establishment ofnew WatsonCrick base pairs in heteroduplex products. However, unlike the initial recognition of homology, strand exchange is stereospecific, requiring the proper antiparallel orientation of complementary strands. Homologous genetic recombination is a reaction of broken DNA molecules: breaks are repaired by aligning complementary bases to restore precisely the integrity of the original DNA sequence. There appear to be two paradigms by which such repair occurs (1). The simpler paradigm involves the splicing of overlapping sequences, in which case rejoining results directly from the renaturation of two complementary single-stranded ends. A second paradigm, revealed by the action ofRecA protein, is more complicated, since homology is recognized not by the pairing of two single strands but rather by the pairing of a single strand with a fully duplex homolog. Recent research has supported the hypothesis that both the recognition of homology and the strand exchange that follows are mediated by triple-helical intermediates (1-7). RecA protein creates such triplex intermediates from nonspecialized sequences, viz., sequences other than homopurine-homopyrimidine tracts, which create a different kind of triplex DNA (8-11). The structure of the triplex intermediate that is produced by RecA protein, which is under active investigation, is not known (1, 12, 13). The starting point for the basic reactions of RecA protein is the right-handed helical nucleoprotein filament that it makes on single-stranded DNA (14, 15). Such a filament will pair with naked complementary single strands or with naked The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. duplex DNA. In the former case, pairing produces heteroduplex DNA that remains in the filament; in the latter case, pairing leads to strand exchange, which leaves heteroduplex DNA in the filament and produces a displaced single strand that is identical in sequence to the (+)-strand contained in the filament (16, 17). Whereas the ultimate formation of stable heteroduplex joints in homologous recombination is indisputably based on Watson-Crick base pairing, the process of recognition and the structure ofthe intermediates are not well understood. In the experiments described below, we used oligonucleotides to examine the determinants of homologous recognition within RecA nucleoprotein filaments.