Elements of Recombination in E. coli, RecA, RecBCD, and Chi
What types of biochemical evidence can be found in support of the mechanisms of recombination discussed in the previous section? His-torically, the elucidation of metabolic pathways was often assisted by the isolation of mutations in enzymes catalyzing individual steps of the pathway. With similar objectives in mind, mutations were isolated that decreased or increased the ability of phage, E. coli, yeast, and some other organisms to undergo recombination. Subsequently, the enzyme prod-ucts of the recombination genes from E. coli and yeast have been identified and purified. Of greatest importance to recombination are the RecA and RecBCD proteins. Additional enzyme activities that might be expected to play roles, such as DNA ligase, DNA polymerases, single-stranded binding protein, and proteins that wind or unwind DNA, have already been discussed and will not be further mentioned here.
RecA mutants are unable to engage in genetic recombination, and asexpected, the purified RecA protein possesses a variety of activities that appear related to recombination. In the presence of ATP the protein binds to single-stranded DNA in a highly cooperative manner. Once one molecule has bound to a stretch of single-stranded DNA, other molecules line up beside it. Then, with the hydrolysis of ATP the protein catalyzes the pairing of a homologous DNA strand from a double-stranded duplex to the strand covered with RecA protein.
Figure 8.16 RecBCD binds to a free end of the DNA and moves at more than200 base pairs per second. As RecBCD crosses a chi site, one of the strands is cut.
The known activities of the RecBCD protein suggest that this protein generates the single-stranded regions that initiate the strand invasion process catalyzed by RecA. RecBCD protein binds at the free end of a double-stranded DNA duplex and moves down the DNA separating the DNA strands and leaving a single-stranded region in its wake. If it encounters a specific eight nucleotide sequence called a chi sequence, the enzyme cleaves one of the strands, leaving a single-stranded DNA end that can be covered with RecA protein and initiate recombination (Fig. 8.16).
Chi can stimulate recombination at distances up to 10,000 bases away from itself and appears to act only in one direction from itself, that is, it possesses a polarity. Only when chi is crossed by RecBCD traveling in one direction does cleavage occur. Apparently organisms other than bacteria utilize sequences that function like chi, since hotspots of genetic recombination are found in yeast and fungi as well. The fact that RecBCD can enter DNA only at an end restricts its stimulation of recombination to those situations in which ends are generated. In addition to random chromosome breakage, in which case genetic re-combination may be a good way to attempt rescue, ends are generated during transfer of genetic information. The rate of genetic recombina-tion between homologous DNA sequences during normal growth is very low. Recombination primarily occurs at the time of introduction of new DNA into bacteria. The analogous principle applies to meiosis of eu-karyotic cells, but the recombination stimulating process there is not yet known.