Site-specific DNA recombination system Min of plasmid p15B: a cluster of overlapping invertible DNA segments.

Abstract

Plasmid pl5B of Escherichia coli 15T-carries a 3.5-kilobase segment that undergoes frequent DNA inversion mediated by the DNA inversion enzyme Min, a member of the Din family of site-specific recombinases. While the previously described Din inversion systems invert a DNA segment between two crossover sites in inverted orientation, the Min system produces more complex DNA rearrangements. These have been physically characterized by electron microscopy and by restriction cleavage analysis. The results can best be explained by a model that involves six crossover sites (called mix) and predicts 240 isomeric forms of the invertible region.The model was confirmed by sequencing the six mix sites in plasmids that contain the invertible DNA segments in a frozen configuration. All mix sites fit the dix consensus sequence, and they are all good substrates for DNA inversion when carried in inverted orientation. Recombination between two mix sites in direct orientation was rare, in line with the notion that Din inversion systems are topologically biased to the inversion reaction. Another recently described multiple inversion system, the shufflon of the E. coli plasmid R64, is neither functionally nor structurally related to the Min system of pl5B.gene also encodes a recombinational enhancer element (unpublished data). In contrast to all previously described Din systems, which invert a DNA segment between two crossover sites, the Min system (Min for multiple DNA inversion) causes more complex DNA rearrangements. The characterization of this mobile DNA region of p15B is the subject of this report.During our studies another example of clustered DNA inversion segments has been described on the R64 plasmid of E. coli (10). In this system, which has been named "shufflon," four DNA segments can invert independently or in groups resulting in complex DNA rearrangements. DNA inversion is brought about by site-specific recombination mediated by the Rci recombinase. Although the Min system of pl5B and the shufflon of R64 show a similar clustering of overlapping invertible DNA segments, they are not related because the DNA sequences of neither the recombinase genes (unpublished data) nor, as we show here, the crossover sites are homologous. In turn, the Rci enzyme shares significant sequence homologies with the recombinases of the integrase family (11).The prokaryotic site-specific DNA recombination systems that have been well characterized so far can be classified into two groups on the basis of their structural and functional homologies and of their mechanistic properties (1,2). One group of loosely related enzymes includes the integrases of phage A (3) and other lambdoid phages, the Cre recombinase of phage P1 (4), and the eukaryotic FLP enzyme of yeast (5).The other group (6) is subdivided into two families: one comprises the resolvases of the Tn3 family of transposons, while the other family consists of a number of DNA invertases (Din family). Site-specific recombinases for the latter are found in the genomes of phages P1 (Ci...

DNA Inversion on Conjugative Plasmid pVT745

“…It was shown in the present study that pDMG27, the pVT745 derivative with an inactivated inv, was still segregationally stable in the original host strain after 200 generations. This result is in line with the notion that recombination between two repeats in direct orientation, which would cause the deletion of the intervening DNA segment, is inefficient in the Din family of inversion systems (24).…”

“…Custom-made oligonucleotide primers were used to sequence the junction fragments. Analysis of the resulting DNA nucleotide sequence revealed the presence of a 22-bp repeat (IR22) with dyad symmetry in opposite orientation at the junctions ( (24), and dix, the consensus sequence for the recombination sites of all members of the Din family (14). Areas of dyad symmetry are indicated by arrows.…”

Genome of Bacteriophage P1

“…Inversions within the analog of the C segment in phages with similar invertible sequences have been reported and may well occur in P1 as well (267). In the p15B plasmid, whose S gene is homologous to that of P1 in its posterior region, inversions can occur at six crossover sites within the corresponding invertible segment and may result in 240 isomeric configurations of this region (269,270). The inversions depend on the p15B invertase Min, which can functionally replace the Cin invertase of P1 (154).…”

Site-specific recombination by the DDE family member mobile element IS30transposase

“…1). In the natural systems, the orientation of recombination sites has similar determinative function (29)(30)(31), although accessory functions have evolved in many cases to restrict the secondary activities [e.g., FIS enhances inversion by Gin-invertase (32)]. …”

“…4B). Truncated IS30 Tpase carries out secondary reactions similarly to natural systems: Serinvertases cause deletion or cointegration (30,31,34) and can integrate into secondary sites (35). More accurate functioning can be achieved by natural selection, as shown in the presented model (Fig.…”