The topology of homologous pairing promoted by RecA protein

Abstract

In addition to catalyzing the pairing of linear single-stranded DNA with homologous duplex DNA, recA protein promotes the pairing of circular single strands with linear duplex DNA or nicked circular duplex DNA, and of gapped circular duplex DNA with superhelical DNA. RecA protein will thus produce joint molecules of DNA at a high frequency from a pair of homologous molecules if one of them is single-stranded or partially single-stranded, and if either one has a free end. The structure made from a linear single strand and duplex DNA is a D loop. The joint molecule made from circular single-stranded DNA and linear duplex DNA is a branched structure in which the circular strand has displaced a strand from one end of the duplex molecule. In these structures, the heteroduplex regions reach sizes approaching that of full-length fd DNA. When we used restriction fragments of duplex fd DNA that were approximately half-length, we found circular molecules that were half duplex and half single-stranded. Similarly, single-stranded circles displaced a strand from nicked circular duplex DNA, yielding structures related to those made with linear duplex DNA, as well as other structures. Our observations indicate that purified recA protein catalyzes a concerted strand transfer with several features of particular biological interest, including the initiation of a strand crossover (in some cases perhaps the crossing back of a strand as well) and the production of long heteroduplex joints by a kind of branch migration. While a free end permits interwinding of DNA strands and the formation of joints containing stable right-handed helices, the free end is not essential for the promotion of homologous pairing by recA protein. When we mixed phage G4 double-stranded DNA and recA protein with single-stranded circular M13 DNA containing an insert of 274 bases of G4 DNA, we observed by electron microscopy the formation of a few percent of complexes in which single-stranded circular DNA and duplex DNA were joined side by side in the region of shared sequence homology. The frequency of such complexes was twenty to thirty times greater than that observed in a control mixture of G4 duplex DNA and single-stranded circular fd DNA, molecules which do not share a region of extensive homology. We conclude that recA protein can promote homologous association of a single strand and duplex DNA without the plectonemic colling that characterizes the normal Watson-Crick structure of DNA.