Intermediates in genetic recombination of bacteriophage T7 DNA

Abstract

DNA molecules formed by interaction of two molecules of bacteriophage T7 DNA were isolated from infected bacteria incubated under conditions which did not permit phage DNA replication. After Escherichia coli 594 su − cells were infected with 32 P and 5-bromodeoxyuridine-labelled T7 phage carrying five amber mutations in genes 1·3, 2, 3, 4 and 5 , intermediate density DNA molecules were isolated by a CsCl equilibrium density-gradient centrifugation. The intermediate density fraction contained dimeric T7 DNA and was further purified by zone centrifugation on a CsCl gradient. The dimeric DNA molecules had “H” or “X” configuration. The lengths of two branches from the same branch point were equal, and the sum of length of one of the branches at each branch point and the length of the region between the two branch points equalled the length of monomeric T7 phage DNA. When doubly branched molecules obtained by co-infection of T7 phage with an Eco RI-sensitive mutation and BrdU-labelled T7 phage without the mutation were digested by Eco RI restriction endonuclease, only one of the four branches of the molecule was cleaved by Eco RI endonuclease. This result indicates that the doubly branched molecules consisted of two parental DNA molecules. By using the Eco RI-cleaved site as a marker, the regions between two branch points were mapped. The regions did not seem to distribute randomly on the molecules. The size of the region varied with the mean length of about 1000 nucleotide pairs. The product of gene 6 , 5′-exonuclease, is required for the formation and/or maintenance of doubly branched molecules. Models of genetic recombination that involve formation of a branched molecule as an intermediate are discussed.