Genetic recombination of bacteriophage T7 DNA in vitro III. A physical assay for recombinant DNA

Abstract

When density-labeled, 32P-labeled T7 DNA is incubated with a T7 +-infected cell extract and the DNA is banded to equilibrium in a CsCl density gradient, a substantial proportion of the 32P label assumes a buoyant density close to that of light T7 DNA. This shift in density is not due to the attachment of lipids or proteins to the DNA, nor is it due to semiconservative replication of the DNA in vitro. On the contrary, the shift in density is believed to be due to recombination between the exogenous DNA and the DNA present in the extract. The [ 32P]DNA is covalently joined as large double-stranded DNA fragments (12,000–18,000 base pairs) to endogenous DNA which sediments as structures two to four times the length of mature T7 DNA. We have termed these molecules recombinant T7 DNA. The formation of recombinant T7 DNA is independent of DNA packaging but requires the active products of T7 genes 1.3 (ligase), 3 (T7 endonuclease 1), 4 (DNA initiation protein), 5 (T7 DNA polymerase), and 6 (T7 exonuclease) to be present during the phage infection. Using a temperature-sensitive mutant, it was demonstrated that the gene 6 exonuclease is acting during the in vitro reaction to promote recombination.