Biologically active recombinant formed through DNA pairing by purified recA protein in vitro.
We have detected in vitro homologous recombination mediated by purified recA protein of Escherichia coli as a recombinant phage produced by using the DNA packaging system of phage lambda. When double-stranded DNA of phage lambda carrying amber mutations is incubated with double-stranded DNA carrying the wild-type genes in the presence of recA protein, Mg++ and ATP, and the DNA packaged, amber+ recombinant phage is produced at a high frequency. This reaction depends completely upon the function of the wild-type recA protein. After incubation of 32P-labeled linear DNA (Form III) with bromouracil-labeled circular DNA (Form I-Form II mixture) in the presence of recA protein, Mg++ and ATP, about 10% of the 32P-counts band at an intermediate density in CsCl equilibrium gradient. This fraction yields a high percentage of the recombinant phage after DNA packaging and shows the alpha-shaped and sigma-shaped joint molecules of linear and circular DNA under the electron microscope. Furthermore, we demonstrate that a non-homologous region inhibits the recombination reaction when it is between the marker concerned and the closer cos end. Our results indicate that recA protein acts directly in the initial step of recombination to join the homologous double-stranded DNA and that the resulting molecule can be matured into the recombinant DNA.
D001426:Bacterial Proteins / Q000235:genetics*; D010582:Bacteriophage lambda / Q000235:genetics; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D009690:Nucleic Acid Conformation; D011938:Rec A Recombinases; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D011938:Rec A Recombinases