Recombination in recA cells between direct repeats of insertion element IS1
Journal of Bacteriology
The ISI sequences that flank the Tn9 chloramphenicol acetyltransferase gene as direct repeats recombine after transformation into an Escherichia coli recA strain. The recombination requires the A PL promoter on the plasmid. A plasmid that contains mutant IS] elements does not recombine. These results indicate that this recombination requires an IS1-specific gene product. The recombinational activity of IS) may resolve transient cointegrates formed during the transposition of IS). I discuss a
... IS). I discuss a possible role for the A PL promoter. Insertion sequences are mobile genetic elements that translocate to new positions in the same or different genomes. In addition, insertion sequences catalyze DNA rearrangements in their vicinity including deletions (8, 34) and inversions (8). The insertion sequence IS] is a 768-base-pair transposable sequence (18, 27) that transposes by a recA-independent mechanism to a large number of sites. The transposition of Tn3 from one replicon to another occurs in two steps (2, 15, 17, 35) . The first step is the generation of a cointegrate intermediate that contains direct repeats of Tn3. The second step is a site-specific recombination between the Tn3 elements that resolves the cointegrate intermediate. This step generates the target replicon containing an insertion of Tn3 and regenerates the original donor replicon. The resolution reaction is catalyzed by the tnpR polypeptide encoded by Tn3 (17, 19, (29) (30) (31) . Although the results of experiments with Tn3 have provided models for transposition which require a cointegrate intermediate, it is not clear whether other transposons and insertion sequences also require cointegrate intermediates for their transposition. For example, Galas and Chandler (14) concluded that IS] either transposes directly or forms cointegrates that are not transposition intermediates. They base their conclusion on the observation that ISI-generated cointegrate molecules are formed at a frequency of 10% of the transposition frequency, and that cointegrate molecules, once formed, do not readily resolve. They reasoned that, due to their stability, if cointegrates were transposition intermediates, then their frequency would have to be much higher to account for the transposition frequency. In contrast, the results of Reif and Arber (33) suggest that ISI normally transposes via a cointegrate intermediate. They cloned a kanamycin resistance gene into the single IS] PstI site. The mutant (ISf kan) exhibited a wild-type transposition frequency in rec+ cells, but not in recA-cells. In the rec-cells, IS] transposition was decreased 100-fold, whereas the frequency of cointegrate formation was elevated to levels comparable to the transposition frequency in rec+ cells. Cointegrates were rare in rec+ cells. This result suggests that the recA gene product has the ability to resolve ISI kan cointegrates and can therefore substitute for a resolvase activity that is supplied by IS], but not the mutant ISl kan. The results presented in this paper support the results of Reif and Arber (33). Tn9 consists of a sequence that encodes resistance to t Present address: Immunex Corp., Seattle, WA 98101. chloramphenicol (cat) flanked by direct repeats of ISI (1, 21, 32). There are three long open reading frames that I have designated A, B, and C (Fig. 1) . Machida and co-workers (22) suggest that the A and B reading frames (which they designate ins a and ins b) encode polypeptides that are required for ISl transposition. The IS] elements of Tn9 recombine in a recA-strain under some conditions. Since the IS1s of Tn9 are directly repeated, they resemble an IS1-generated cointegrate, and the recombination between the direct repeats of IS1 of Tn9 resembles a resolution reaction similar to that observed for Tn3 (2, 15, 17, 35) . The results of experiments presented here suggest that an ISf gene product is required for the recombination between direct repeats of ISf that are formed during the transposition of ISf. MATERIALS AND METHODS Bacterial strains, plasmids, and phages are listed in Table 1 . A recA::TnlO allele that has an insertion of TnlO into the recA gene (Gellert, unpublished data) was introduced into N721 and N1106 by P1 transduction with P1 cm cIrlOO (26) grown on N1680. Transductants were found by selection for resistance to 25 ,ug of tetracycline per ml. Tetracycline-resistant cells were tested for sensitivity to UV light. Lambda lysogens were constructed by incubating cells at 37°C for 20 min with A at a multiplicity of infection of 1 before plating on LB plates (26) seeded with K b 2 c to select for A immunity. The resulting strains are N1694 and N1713. Manipulation of DNA. Plasmid extraction was by the method of Birnboim and Doly (5). Restriction endonucleases, ligase, and polymerase were purchased from either New England Biolabs or Bethesda Research Laboratories. Restriction enzyme digests were done as recommended by the suppliers. The conditions for DNA polymerase I large fragment were as described previously (23) . Ligations were done in a 100-,ul volume containing 10 mM Tris (pH 7.4), 10 mM dithiothreitol, 10 mM MgC92, 10 mM ATP, and one Weiss unit (37) of T4 DNA ligase. Ligations were done at 14°C for at least 1 h or at room temperature overnight. Electrophoresis of DNA was on 0.7% agarose gels in Tris-acetate buffer (23) or on 0.5% acrylamide gels in TBE (23). DNA was extracted from agarose gels by the "freezesqueeze" method of Thuring and co-workers (36), except that 10 pug of tRNA per ml was added to the gel extract to insure quantitative precipitation of small amounts of DNA.