SUMMARYGin-mediated site-specific recombination promotes inversion of the G segment of phage Mu. The crossover takes place between two 34 bp-long inverted repeat sequences flanking the G segment. We have characterized the inversion site, the target for the site-specific recombination mechanism. An artificial invertible segment was constructed which consists of parts of the invertible segments of Mu and phage P1, which in this respect are largely homologous, Upon inversion of this hybrid segment the crossover site could be located, by DNA sequencing, in the ACCT sequence of the centre of symmetry in the inverted repeat in Mu. The hybrid Mu-P1 segment inverts at a lower frequency than its parental invertible segments probably because of the mismatches between the inverted repeats of Mu and P 1. This suggests that base pairing between the inverted repeats is an intermediate step in recombination. Plasmids with subcloned G segments lacking the adjacent/~ region of Mu or the corresponding region in P7, a relative of P1, are deficient in inversion. By analysis through site-specific mutagenesis of Mu DNA, an enhancer element with multiple recognition sites was identified which is necessary for efficient inversion. This component of the inversion site was located in a 170 bp segment within the Mu/~ region, 30 bp to the right of the inverted repeat sequence, but can be separated from the crossover site by a 1200 bp insertion without losing its effect.
We have determined the nucleotide sequence of the cin gene of phage P7, which encodes a DNA inversion function with a predicted molecular weight of 21241T. The sequence of one strand was determined by Maxam and Gilbert sequencing from SphI and KpnI sites in plasmid pWR1 (1,2) and is shown together with the derived aminoacid sequence of cin. As previously described (1,2,3), the location and orientation of the cin gene of P7 with respect to thelljacent invertible C segment are identical to that of the related phage P1. i.e. the COOH-terminal part overlaps the outer end of the inverted repeat (IR [) sequence, which is the substrate for the cin-promoted recombination. Comparison with the DNA sequence of P1 (4) shows that the cin genes are 96% idenrtlal, and only altered nucleotides in P1 are shown. Three exchanges with respect to te aminoacid sequence do not affect the consensus sequence of the family of DNA invertases (5), so that we assume that P7 Cin is structurally and functionally indistinguishable from P1 Cin. In contrast to the well conserved coding region, the 5' upstream sequence is more diverged except for a "-10' like sequence (boxed), which therefore could be part of the promoter used in both systems. These potential Prlbnow boxes are located in the left arm of a synnetrical AT-rich sequence (<-+->), which could well fit a recognition site for a regulatory protein of yet unknown origin and function.
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