Phage recombinases and their applications

The homologous recombination systems of linear double-stranded (ds)DNA bacteriophages are required for the generation of genetic diversity, the repair of dsDNA breaks, and the formation of concatemeric chromosomes, the immediate precursor to packaging. These systems have been studied for decades as a means to understand the basic principles of homologous recombination. From the beginning, it was recognized that these recombinases are linked intimately to the mechanisms of phage DNA replication. In the last decade, however, investigators have exploited these recombination systems as tools for genetic engineering of bacterial chromosomes, bacterial artificial chromosomes, and plasmids. This recombinational engineering technology has been termed “recombineering” and offers a new paradigm for the genetic manipulation of bacterial chromosomes, which is far more efficient than the classical use of nonreplicating integration vectors for gene replacement. The phage λ Red recombination system, in particular, has been used to construct gene replacements, deletions, insertions, inversions, duplications, and single base pair changes in the Escherichia coli chromosome. This chapter discusses the components of the recombination systems of λ, rac prophage, and phage P22 and properties of single-stranded DNA annealing proteins from these and other phage that have been instrumental for the development of this technology. The types of genetic manipulations that can be made are described, along with proposed mechanisms for both double-stranded DNA- and oligonucleotide-mediated recombineering events. Finally, the impact of this technology to such diverse fields as bacterial pathogenesis, metabolic engineering, and mouse genomics is discussed.