The double-strand break that precedes recombination must be processed before recombination can take place. This processing is performed by the RecBCD protein complex, which cuts back or resects the double-strand break DNA end. RecB and recC mutants were isolated in initial screens for conjugation-defective and UV-sensitive E. coli mutants. Later studies showed that the RecB and Recc proteins act in a complex that also contains the ReeD protein. The RecBCD complex has several biochemical activities a 3'→5'exonuclease activity, a 5'→3'exonuclease activity, and a DNA helicase activity. These three activities work together to prepare the double-strand break end for HR. The RecBCD complex binds to double-strand break ends and then unwinds the double strand through the helicase activity while degrading each single strand separately. Degradation of two single strands occurs at different rates, with the 3'→5'exonuclease activity being predominant, so that the 3' end is degraded more rapidly. This degradation pattern continues until the RecBCD complex reaches a sequence called a chi site.
Chi sites were discovered by investigators who were studying phage mutants with defects in their recombination system. These recombination-deficient (red) mutants depend on the bacterial RecBCD complex to perform the essential recombination functions needed for viral DNA replication. Because the bacterial recombination system is a poor substitute for the phage system, bacterial infection produces small plaques. However a few large plaques sometimes are formed. Lambda phages isolated from these large plaques were shown to have an additional mutation that created hot spots (now called chi sites), which allowed the bacterial RecBCD complex to function more efficiently.
Subsequent studies showed that cbi sites have the sequence 5'-GCTGGTGG-3'and are quite common inthe bacterial genome, occurring about once in every 5000 bp in the E.coli genome (a much higher frequency than would be predicted for a random nucleotide sequence). When the RecBCD complex encounters a cbi site, its 3'→5' exonuclease activity decreases, while its 5'→3' exonuclease activity increases, leading to the formation of a 3'single-strand overhang. RecA is loaded by the RecBCD complex and binds to the overhang to form a nucleofilament, facilitating its invasion into the homologous duplex and initiating HR. Because the chi site is not symmetric, it stimulates recombination in only one direction.