POINT MUTATIONS IN THE INTEGRON INTEGRASE INTI1 THAT AFFECT RECOMBINATION AND OR SUBSTRATE RECOGNITION/

The site-specific recombinase IntI1 found in class 1 integrons catalyz es the excision and integration of mobile gene cassettes, especially a ntibiotic resistance gene cassettes, with a site-specific recombinatio n system. The integron integrase belongs to the tyrosine recombinase ( phage integrase) family. The members of this family, exemplified by th e lambda integrase, do not share extensive amino acid identities, but three invariant residues are found within two regions, designated box I and box II. Two conserved residues are arginines, one located in box I and one in box II, while the other conserved residue is a tyrosine located at the C terminus of box II. We hare analyzed the properties o f IntI1 variants carrying point mutations at the three conserved resid ues of the family in in vivo recombination and in vitro substrate bind ing. We have made four proteins with mutations of the conserved box I arginine (R146) and three mutants with changes of the box II arginine (R280); of these, MBP-IntI1(R146K) and MBP-IntI1 (R280K) bind to the a ttI1 site in vitro, but only MBP-IntI1(R280K) is able to excise casset tes in vivo. However, the efficiency of recombination and DNA binding for MBP-IntI1(R280K) is lon er than that obtained with the wild-type M BP-IntI1. We have also made two proteins with mutations of the tyrosin e residue (Y312), and both mutant proteins are similar to the wild-typ e fusion protein in their DNA-binding capacity but are unable to catal yze in vivo recombination.