STRUCTURE AND FUNCTION OF 59-BASE ELEMENT RECOMBINATION SITES ASSOCIATED WITH MOBILE GENE CASSETTES

The integration of gene cassettes into integrons is effected by site-s pecific recombination catalysed by an integrase, Intl, encoded by the integron. The cassette-associated recombination sites, 59-base element s, are not highly conserved and vary in length from 57 to 141 bp, They can be identified by their location and the relationship of over 20 b p at their outer ends to consensus sequences that are imperfect invert ed repeats of one another. The recombination cross-over occurs close t o one end of the 59-base element, within a conserved core site with th e consensus sequence GTTAGGC or GTTRRRY. By introducing single-base ch anges at each of these positions in the aadB 59-base element, bases th at are critical for site activity were identified, The recombination c ross-over was also localized to a unique position between the adjacent G and T residues. Changes introduced in the conserved AAC of the inve rse core site (GCCTAAC or RYYYAAC) located at the opposite end of the 59-base element also reduced site activity but to a lesser extent. Seq uences of rare recombinants revealed an alternative position for stran d exchange and led to the conclusion that 59-base elements comprise tw o simple sites, analogous to those recognized by other integrases, wit h each simple site made up of a pair of inversely oriented Intl bindin g domains separated by a spacer of 7 or 8 bp. Re-examination of the se quences of all known 59-base elements revealed that this simple site c onfiguration was present at bath the left and right ends in all 59-bas e elements, The identity of bases in the spacer is not required for ef ficient recombination and the cross-over is located at one end of the spacer, suggesting that during Intl1-mediated recombination only one s trand exchange occurs.