Intermediates in genetic recombination of bacteriophage T7 DNA. Biological activity and the roles of gene 3 and gene 5

Abstract

When Escherichia coli cells were infected with 32P- and 5-bromodeoxyuridine-labeled T7 bacteriophage defective in genes 1.3, 2.3, 4 and 5, doubly branched T7 DNA molecules with “H” or “X”-like configurations were found in the half-heavy density fractions. Physical study showed that they are dimeric molecules composed of two parental DNA molecules (Tsujimoto & Ogawa, 1977 a). The transfection assay of these molecules revealed that they were infective. Genetic analysis of progeny in infective centers obtained by transfection of dimeric molecules formed by infection of genetically marked T7 phage showed that these dimeric molecules were genetically biparental. To elucidate the roles of the products of gene 3 (endonuclease I) and gene 5 (DNA polymerase) of phage T7 in the recombination process, the 32P/BrdUrd hybrid DNA molecules which were formed in the infected cells in the presence of these gene products were isolated, and their structures were analyzed. The presence of T7 DNA polymerase seems to stimulate and/or stabilize the interaction of parental DNAs. At an early stage of infection few dimeric molecules were formed in the absence of T7 DNA polymerase, whereas a significant number of doubly branched molecules were formed in its presence. With increasing incubation time, the multiply branched DNA molecules with a high sedimentation velocity accumulated. In contrast to the accumulation of multiply branched molecules in phage with mutations in genes 2, 3 and 4, almost all of the 32P/BrdUrd hybrid DNA formed in phage with mutations in genes 2 and 4 were monomeric linear molecules. Shear fragmentation of monomeric linear 32P/BrdUrd-labeled DNA shifted the density of [ 32P]DNA to almost fully light density. It was also found that approximately 50% of [ 32P]DNA was linked covalently to BrdUrd-labeled DNA. These linear monomer DNA molecules had infectivity and some of those formed by infection of genetically marked parents yielded recombinant phages. Therefore the gene 3 product seems to process the branched intermediates to linear recombinant molecules by trimming the branches.