There are some other attributes of Rec A, which make its role in recombination understandable. These include the following : (i) ability to polymerize single strands; (ii) ability to incorporate duplex DNA into filaments, giving duplexes longer than the original duplex. In view of this, the reaction involving generation of heteroduplexes during recombination may involve following three steps : (i) a slow presynaptic phase, in which Rec A polymerizes on single stranded DNA; (ii) a fast pairing reaction between single stranded DNA and its complement in double stranded DNA, to produce heteroduplex DNA; (iii) a slow displacement of one strand from the duplex, to produce a long region of heteroduplex DNA. The initial region of heteroduplex DNA may not be found in a double helical form (plectonemic),
but the two strands may be associated side by side (paranemic joint).
This paranemic joint is unstable and further progress in recombination events requires its conversion to double helical form.
When the reacting DNA molecules are circular, the product of Rec A sponsored reaction is a chi (χ) structure. Although action of Rec A is independent of DNA sequence, certain hot spots stimulate the Rec A recombination system. These hot spots were discovered in lambda phage chi
mutants and share common non-symmetrical sequence of 8 bp (5'GCT GG TGG3'). This sequence is called chi sequence
and is not necessary for recombination. However, it stimulates recombination in its vicinity and identifies targets for the enzyme exonuclease V synthesized under the control of Rec BCD
genes. This enzyme has following roles : (i) it degrades DNA; (ii) it can unwind duplex DNA
in the presence of SSB; (iii) it has an ATPase activity. Thus it provides a single stranded region with a free end. These free ends are then seized by Rec A protein, which requires free termini to start the strand assimilation reaction.
Recombination, like replication, also requires topological manipulation of DNA, which is facilitated by topoisomerases. These enzymes may relax or introduce supercoils in DNA and are required to disentangle DNA molecules that have become catenated by recombination or replication. Topoisomerases can be of Type I
(making transient break in one strand) or Type II
(making transient double strand breaks). Type I topoisomerases (e.g. product of top A
gene of E. coli
)relax negatively supercoiled DNA, while Type II topoisomerases (e.g. gyrase)
relax both negative and positive supercoils, (for supercoiling consult Chemistry of the Gene 1. Nucleic Acids and Their Structure
). The removal of supercoils from the crossing segments is required during the recombination process.