The RecA protein promotes DNA strand exchange, as shown in Figure 13-11, with a unidirectional branch migration proceeding around the DNA circle. The direction of the branch migration was established by Cox and Lehman. A circular chromosome from bacteriophage ϕX174 can be isolated as either a single-or a double-stranded circle. The circular map of the ϕX174 genome is shown in Figure 1 (data from Cox and Lehman’s paper); the single-stranded circle proceeds 5′→3′ in the clockwise direction. In the experiment, the single-stranded ϕX174 circle was radioactively labeled uniformly along its length. The double-stranded circular DNA was not labeled, but was cleaved at a unique site (labeled A) by a restriction enzyme to generate the substrates shown in Figure 13-11. Sites for cleavage by a second restriction enzyme (labeled B) are also noted.

A DNA strand exchange reaction was initiated with the RecA protein and ATP. At various times, aliquots were removed and treated with restriction enzyme B and with a nuclease that selectively digested all single-stranded DNA, and the DNA was subjected to agarose gel electrophoresis to separate the restriction fragments. The incorporation of radioactivity (as percentage of maximum) into the various restriction fragments was measured at various time intervals; the results are shown in Figure 2.