CATALYTIC RESIDUES OF GAMMA-DELTA RESOLVASE ACT IN CIS

The resolvase protein of the gamma delta transposon is a site-specific recombinase that acts by a concerted break-and-join mechanism. To ana lyse the role of individual resolvase subunits in DNA strand cleavage, we have directed the binding of catalytic mutants to specific recombi nation crossover sites or half-sites. Our results demonstrate that the resolvase subunit bound at the half-site proximal to each scissile ph osphodiester bond provides the Ser10 nucleophile and Arg8, Arg68 and A rg71 residues essential for cleavage and covalent attachment to the DN A. Several other residues near the presumptive active site are also sh own to act in cis. Double-strand cleavage at one crossover site can pr oceed independently of cleavage at the other site, although interactio ns between the resolvase dimers bound at the two crossover sites remai n essential. An appropriately oriented heterodimer of active and inact ive protomers can in most cases mediate either a 'top' or 'bottom' sin gle-strand cleavage, suggesting that there is no obligatory order of s trand cleavages. Top-strand cleavage is associated with the topoisomer ase I activity of resolvase, suggesting that a functional asymmetry ma y be imposed on the crossover site by the structure of the active syna pse.