The effect of nonhomologous DNA sequences on interplasmidic recombination

The effect of nonhomologous DNA sequences at one or both sides of short genetic intervals on recombination within that interval was investigated, using an interplasmidic recombination system in Escherichia coli K-12. The recombining plasmids were derivatives of pBR322 and pACYC184, which share a 1330-nucleotide sequence that includes the tet gene. The genetic interval was defined by the HindIII and BamHI or BamHI and the SalI restriction endonuclease sites of this gene. The substantial differences between recombination frequencies measured within intervals bracketed or bounded on one side by major nonhomologies suggests that, in this system, strand exchange is polar and is blocked by major nonhomologies. This conclusion is substantiated by results of three-factor crosses and by structural analysis of recombination products. Results of two-factor crosses in recA genetic background and structural analysis of recombination products suggest that strand exchange occurs in the absence of a functional recA gene. "Opening" a bracketed BamHI-SalI genetic interval of the tet gene, at the SalI site, by substituting a major insertion with a short deletion, results in an increase in recombination frequency, within this genetic interval, which is greater than expected on the basis of the ratio of the length of homology on the two sides of the SalI site. This observation suggests that a genetic element that may affect rate of recombination initiation, polarity of strand exchange, template specificity in mismatch repair or more than one of these events may be present on the outer side of the SalI site of the tet gene.