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Genetics, Vol 134, 1013-1021, Copyright © 1993
INVESTIGATIONS |
Efficient Double-Strand Break-Stimulated Recombination Promoted by the General Recombination Systems of Phages {lambda} and P22
A. R. Poteete and A. C. Fenton
Department of Molecular Genetics and Microbiology, and Program in Molecular Medicine, University of Massachusetts, Worcester, Massachusetts 01605
To examine bacteriophage recombination in vivo, independent of such other processes as replication and packaging, substituted {lambda} phages bearing restriction site polymorphisms were employed in a direct physical assay. Bacteria were infected with two phage variants; DNA was extracted from the infected cells and cut with a restriction endonuclease. The production of a unique recombinant fragment was measured by Southern blotting and hybridization with a substitution sequence-specific probe. High frequency recombination was observed under the following conditions: the substituted {lambda} phages infected a wild-type host cell bearing a {lambda} repressor-expressing plasmid designed to shut down phage transcription and inhibit phage DNA replication as well. The same plasmid expressed the {lambda} red and gam genes. In addition, the host cell bore a second plasmid which expressed the EcoRI restriction-modification system. Both phage chromosomes possessed a single EcoRI site in the middle of the marked substitution sequence; however, as the site was modified in one of the parent phages, only the other partner was cut. Recombination was found to be dependent upon (1) red, (2) recA, (3) inactivation of the host recBCD function, either by Gam protein or by mutation and (4) double-strand breaks. The homologous recombination system of phage P22 could substitute for that of {lambda}.
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