Enzymology of Genetic Recombination

Abstract

Studies of genetic recombination in prokaryotes have shown (1) that recombination occurs by breakage and reunion of DNA, sometimes, but not always, associated with new DNA synthesis, and (2) that the parental contributions to a recombinant molecule are commonly joined by a short heteroduplex or hybrid region. In the past few years, some of the enzymes involved in recombination in prokaryotes have been identified, such as the exonuclease made by bacteriophage λ. Recent studies of λ exonuclease make it possible to rationalize most of the properties of the enzyme in terms of its role in producing a perfect heteroduplex joint between homologous molecules of DNA. λ exonuclease cleaves 5′ mononucleotides from the 5′ end of native DNA in a processive fashion, extensively degrading any molecule of DNA before detaching and attacking another molecule of substrate. The latter property suggests that some control prevents the enzyme from playing an exclusively degradative role. 5′ phosphoryl termini located at gaps in one strand of duplex DNA are resistant to the enzyme. Although 5′ phosphoryl termini at nicks are even more resistant, the enzyme appears to bind weakly at such sites. The significance of these properties may be seen in the enzyme's action at the site of a redundant single stranded branch, such as one might expect to find at a joint between two fragments of DNA. A redundant strand is assimilated into the helix, behind λ exonuclease, as the enzyme processively degrades the homologous helical strand. The enzyme recognizes the presence of the redundant strand both for initiation and termination of hydrolysis. Removal of the redundant single strand by the prior action of exonuclease I blocks the action of λ exonuclease on the helical strand. Moreover, when a redundant strand has been completely assimilated through the action of λ exonuclease, the enzyme stops at the precise point which permits the interrupted polynucleotide strand to be sealed by polynucleotide ligase. The sequential action of λ exonuclease and polynucleotide ligase on redundant joint molecules of λ DNA produces intact polynucleotide strands that are biologically active. Several models have been suggested to relate the assimilation of single strands to the genetic recombination of λ and possibly to recombination in other systems as well. Molecules of DNA with double-stranded branches have also been synthesized to test one of the models. The models suggest that λ exonuclease may catalyze a concerted reaction that (1) exposes complementary nucleotide sequences, (2) forms or extends the heteroduplex region, and (3) eliminates redundant branches, precisely restoring a duplex structure that can be sealed covalently by polynucleotide ligase. The λ enzyme, and similar exonucleases, might drive otherwise reversible interactions of a single strand with a recipient duplex, including certain kinds of interactions between two molecules of double-stranded DNA.