Purification and characterization of the R64 shufflon-specific recombinase.

The shufflon, a multiple DNA inversion system in plasmid R64, consists of four invertible DNA segments which are separated and flanked by seven 19-bp repeat sequences. The product of a site-specific recombinase gene, rci, promotes site-specific recombination between any two of the inverted 19-bp repeat sequences of the shufflon. To analyze the molecular mechanism of this recombination reaction, Rci protein was overproduced and purified. The purified Rci protein promoted the in vitro recombination reaction between the inverted 19-bp repeats of supercoiled DNA of a plasmid carrying segment A of the R64 shufflon. The recombination reaction was enhanced by the bacterial host factor HU. Gel electrophoretic analysis indicated that the Rci protein specifically binds to the DNA segments carrying the 19-bp sequences. The binding affinity of the Rci protein to the four shufflon segments as well as four synthetic 19-bp sequences differed greatly: among the four 19-bp repeat sequences, the repeat-a and -d sequences displayed higher affinity to Rci protein. These results suggest that the differences in the affinity of Rci protein for the 19-bp repeat sequences determine the inversion frequencies of the four segments.

Analysis of DNA inversions in the shufflon of plasmid R64.

The shufflon, a multiple DNA inversion system in the plasmid R64, consists of four DNA segments flanked and separated by seven 19-bp repeat sequences. Site-specific recombinations mediated by the rci product occur between each inverted repeat sequence, resulting in inversions of the four segments independently or in groups. The seven 19-bp repeat sequences are classified into four types (repeat-a, -b, -c, and -d), according to their 3-bp variable sequences. We individually cloned A, B, and C segments of the R64 shufflon and determined the in vivo inversion frequency of each segment. The inversion frequencies of three segments differed greatly. The inversion frequency declined in the following order: segments A, B, and C. Synthetic 19-mer oligonucleotides corresponding to both strands of repeat-a, -b, -c, and -d sequences were inserted into appropriate sites of pBR322. The rci-mediated DNA inversion occurred between two synthetic inverted repeats, indicating that the 19-bp inverted repeat sequences are the sole elements required in cis for the shufflon system. The inversion frequencies of DNA segments flanked by various sequences indicate that the four types of repeat sequences determine the inversion frequency of the four DNA segments of the R64 shufflon. Deletion of a DNA segment flanked by direct repeat sequences could not be detected.