Illegitimate recombination mediated in vitro by DNA gyrase of Escherichia coli: structure of recombinant DNA molecules.
We have developed a cell-free system from Escherichia coli for studying illegitimate recombination between nonhomologous DNA molecules. The recombination is stimulated by oxolinic acid, an inhibitor of DNA gyrase. The stimulation is abolished by coumermycin A1 and is not found in extracts of nalidixic acid-resistant (gyrA) mutants. We therefore inferred that DNA gyrase directly participates in illegitimate recombination, at least in the presence of oxolinic acid [Ikeda, H., Moriya, K. & Matsumoto, T. (1981) Cold Spring Harbor Symp. Quant. Biol. 45, 399--408]. The structure of recombinant DNA molecules formed in the presence of oxolinic acid from a cross between phage lambda and plasmid pBR322 DNAs was analyzed by heteroduplex mapping. Among nine isolates tested, two recombinants were formed by the insertion of the plasmid into the lambda genome. The seven other recombinants had more complicated genome structures. Insertion of pBR322 was accompanied by a deletion on one of the genomes. In all cases, the end points of deletions coincided with one end of the pBR322 insertion. Recombination sites seemed to be distributed randomly on the lambda and pBR322 genomes. Analysis of nucleotide sequences of the recombination junctions proved that the crossover took place between nonhomologous DNA sequences. A model for DNA gyrase-mediated illegitimate recombination is discussed.
D010582:Bacteriophage lambda; D001483:Base Sequence; D002474:Cell-Free System; D003001:Cloning, Molecular; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D010093:Oxolinic Acid / Q000494:pharmacology; D010957:Plasmids; D011995:Recombination, Genetic / Q000187:drug effects
D004250:DNA Topoisomerases, Type II; D010093:Oxolinic Acid