COMMUNICATION BETWEEN HIN RECOMBINASE AND EIS REGULATORY SUBUNITS DURING COORDINATE ACTIVATION OF HIN-CATALYZED SITE-SPECIFIC DNA INVERSION

The Hin DNA invertase becomes catalytically activated when assembled i n an invertasome complex containing two Fis dimers bound to an enhance r segment. The region of Fis responsible for transactivation of Hin co ntains a mobile P-hairpin arm that extends from each dimer subunit. We show here that whereas both Fis dimers must be capable of activating Hin, Fis heterodimers that have only one functional activating p-arm a re sufficient to form catalytically competent invertasomes. Analysis o f homodimer and heterodimer mixes of different Hin mutants suggests th at Fis must activate each subunit of the two Hin dimers that participa te in catalysis. These experiments also indicate that all four Hin sub units must be coordinately activated prior to initiation of the first chemical step of the reaction and that the process of activation is in dependent of the catalytic steps of recombination. We propose a molecu lar model for the invertasome structure that is consistent with curren t information on protein-DNA structures and the topology of the DNA st rands within the recombination complex. In this model, a single Fis ac tivation arm could contact amino acids from both Hin subunits at the d imer interface to induce a conformational change that coordinately pos itions the active sites close to the scissile phosphodiester bonds.