The Gin function of bacteriophage Mu catalyzes inversion of the G DNA segment, thus switching the host range of Mu phage particles. This site-specific recombination event takes place between inverted repeat sequences (IR) that border the G segment. Sequences in the Mu beta region extending approximately from position 118 to 178 are essential for efficient inversion. In cis this region, termed sis, stimulates inversion about 15-fold. Neither the relative orientation of sis with respect to the IR sequences nor the distance to IR substantially influences the stimulatory effect. For full activity purified Gin protein must be supplemented with crude host factor from E. coli K12. We suggest that, in addition to Gin, a DNA-binding host protein is required for efficient G inversion.

The DNA invertase Gin encoded by bacteriophage Mu catalyses efficient site‐specific recombination between inverted repeat sequences (IR) in vivo and in vitro in the presence of the host factor FIS and the recombinational enhancer. We demonstrate that Gin alone is able to introduce single strand breaks into duplex DNA fragments which contain the IR sequence. Strand cleavage is site‐specific and can occur on either strand within the IR. Cleaved molecules contain Gin covalently attached to DNA. The covalent complex is formed through linkage of Gin to the 5′ DNA phosphate at the site of the break via a phosphoserine. Extensive site‐directed mutational analysis showed that all mutants altered at serine position 9 were completely recombination deficient in vivo and in vitro. The mutant proteins bind to DNA but lack topoisomerase activity and are unable to introduce nicks. This holds true even for a conservative amino acid substitution at position 9. We conclude that serine at position 9 is part of the catalytic domain of Gin. The intriguing finding that the DNA invertase Gin has the same catalytic center as the DNA resolvases that promote deletions without recombinational enhancer and host factor FIS is discussed.

Site‐specific DNA inversion in phage Mu is catalysed by the phage‐encoded DNA invertase Gin and a host factor FIS. We demonstrate that purified Gin protein binds specifically to 34‐bp sequences that flank the G segment as inverted repeats. Each inverted repeat (IR) contains two binding sites for Gin which have to be arranged in a specific configuration to constitute a recombinogenic site. While one of these sites is bound when present alone, the other site is bound only in conjunction with the first one, suggesting cooperative binding. In addition to the sites within the IR, Gin binds with lower affinity to AT‐rich sequences adjacent to the IR. We demonstrate that these sites do not participate in the inversion reaction. The IR itself can be shortened to 25 bp without effect on inversion frequency. Using gel mobility shift experiments on circular permuted fragments containing the IR we show that Gin bends DNA upon binding. We discuss the possibility that DNA bending is related to the formation of a productive synaptic complex.

Inversion of the G segment in bacteriophage Mu DNA occurs by a site‐specific recombination event and determines the host specificity of Mu phage particles produced. Inversion is mediated by a Mu function (Gin). The gin gene has been placed under control of the inducible λ pL promoter and a synthetic Shine‐Dalgarno linker upstream of the initiation codon. The Gin protein content in induced cells is boosted to ˜10% of total protein. Partially purified extracts from overproducing strains promote efficient inversion of the G DNA segment in vitro which is visualized by agarose gel electrophoresis of the substrate DNA after cutting with appropriate restriction endonucleases. The in vitro reaction requires Mg2+, a super‐coiled DNA substrate and occurs in the absence of exogenous ATP. Inversion from the G(+) to the G(−) orientation is as efficient as the switch from G(−) to G(+).

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