The initial steps in finding enzymes that carry out recombination were genetic screens for mutants of E. colithat are defective in recombination. Assays were developed to test for recombination, and mutants that showed a decrease in recombination frequency were isolated. These were assigned to complementation groups called recA, recB, recC, recD, and so forth. Roughly 20 different genes (different reccomplementation groups) have been identified in E. coli. Each gene encodes an enzyme or enzyme subunit required for recombination.
Many of these genes have been cloned and their encoded products characterized in terms of a variety of enzymatic functions. However, we still do not have a clear picture of how all these enzymes work together to carry out recombination, nor has recombination has been reconstituted in vitrofrom purified components. Further complicating matters is the presence of multiple pathways for recombination. Much work remains to be done to completely understand recombination at a biochemical level. Despite this, the array of recombination enzymes gives us at least a partial view of the mechanisms of recombination. Also, the enzymes characterized in E. coli have homologs and counterparts in other species. Some aspects of the recombination machinery appear to be conserved across a wide phylogenetic range.
The major enzymatic steps are outlined in Fig. 8.12. Three different pathways have been characterized that differ in the steps used to generate the invading single strand of DNA. All three pathways use RecA for homologous pairing and strand exchange, RuvA and RuvB for branch migration, and RuvC and DNA ligase for resolution. These steps and enzymes will be considered individually in the following sections.
Figure 8.12. Enzymatic Steps in Recombination. Three pathways for recombination are shown, starting with a covalently closed, supercoiled circle (with each strand of the duplex shown as a thin line) and a linear duplex (with each strand shown as a thick white line) as the substrates. The three pathways differ in the enzymes used for initiation, but subsequent steps use enzymes common to all three. Adapted from Kowalczykowski, et al. (1994) Microbiological Reviews, 58:401-465.