Gin-mediated recombination of catenated and knotted DNA substrates: implications for the mechanism of interaction between cis-acting sites

The Gin DNA-inversion system of bacteriophage Mu normally requires a substrate containing two inverted recombination sites (gix) and an enhancer sequence on the same supercoiled DNA molecule. The reaction mechanism was investigated by separating these sites on catenated rings. Catenanes with the gix sites on one circle and the enhancer on the other recombined efficiently. Thus, the enhancer was fully functional even though it was located in trans to the gix sites. Multiple links between the rings are required for recombination. Multiply linked catenanes with gix sites on separate circles, one of which contained the enhancer, were also efficient substrates. Knotted constructs carrying directly repeated gix sites were recombined. Catenated and knotted substrates must also be supercoiled. These experiments eliminate simple tracking or looping models as explanations for why the enhancer and gix sites must be in cis with standard substrates. Rather, the Gin synaptic complex requires the three sites to be mutually intertwined in a right-handed fashion with a unique polarity of the gix sites. This geometry is achieved by branching of the DNA substrate and requires the energy and structure of supercoiling, catenation, or knotting.