A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective prophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42°C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries. DNA engineering is conducted routinely in Escherichia coli, not only for genetic studies in bacteria, but also for constructing DNA molecules to be used in studies of other organisms. Most cloning methods use restriction endonuclease cleavage followed by DNA joining with DNA ligase. These in vitro cleavage and joining reactions are the basis for creating DNA recombinants and for cloning of DNA segments on plasmid vectors in which additional modification and amplification can take place. In the yeast Saccharomyces cerevisiae, strategies that generate novel DNA junctions exploit homologous recombination (1). The DNA double-strand break and repair recombination pathway is very efficient in yeast. Functions in this pathway recombine transformed linear DNA with homologous DNA in the yeast cell. Moreover, this recombination is proficient with short synthetic oligonucleotides, thereby permitting recombinant DNA to be generated in vivo without using DNA restriction and ligation (2-4). However, subsequent manipulation of recombinant DNA molecules in yeast, as opposed to E. coli, is laborious, especially if it is to be used for recombinant DNA work in other organisms.Unlike yeast, E. coli is not readily transformed by linear DNA fragments due in part to the rapid degradation of the DNA by the intracellular RecBCD exonuclease (5). Mutant recBCD strains lacking the exonuclease do not rapidly degrade linear DNA; however, such strains are extremely poor growing, are defective for recombination, and do not support efficient replication of the many plasmids used in recombinant DNA work. In certain recA ϩ backgrounds, the recBCD defect for recombination is suppressed, allowing linear DNA to be taken up by the cell and to be recombined (6). Other homologous recombination strategies in E. coli recBC derivatives hav...

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Homologous DNA recombination is a fundamental, regenerative process within living organisms. However, in most organisms, homologous recombination is a rare event, requiring a complex set of reactions and extensive homology. We demonstrate in this paper that Beta protein of phage generates recombinants in chromosomal DNA by using synthetic single-stranded DNAs (ssDNA) as short as 30 bases long. This ssDNA recombination can be used to mutagenize or repair the chromosome with efficiencies that generate up to 6% recombinants among treated cells. Mechanistically, it appears that Beta protein, a Rad52-like protein, binds and anneals the ssDNA donor to a complementary single-strand near the DNA replication fork to generate the recombinant. This type of homologous recombination with ssDNA provides new avenues for studying and modifying genomes ranging from bacterial pathogens to eukaryotes. Beta protein and ssDNA may prove generally applicable for repairing DNA in many organisms.