Specificity determinants for bacteriophage Hong Kong 022 integrase: analysis of mutants with relaxed core-binding specificities

The integrase (Int) proteins encoded by bacteriophages HK022 and lambda cat alyse similar site-specific integration and excision reactions between spec ific DNA regions known as attachment (att) sites. However, the Int proteins of HK022 and lambda are unable to catalyse recombination between non-cogna te att sites. The att sites of both phages contain weak binding sites for I nt, known as 'core-type' sites. Negatively acting nucleotide determinants a ssociated with specific core sites (lambda B', HK022 B', HK022 C) are respo nsible for the barrier to non-cognate recombination. In this study, we used challenge phages to demonstrate that the lambda and HK022 Ints cannot bind to core sites containing non-cognate specificity determinants in vivo. We isolated mutants of the HK022 Int, which bind the lambda B' core site. Two mutants, D99N and D99A, have changed a residue in the core-binding (CB) dom ain, which may be directly contacting the core site DNA. We suggest that bi nding to the lambda B' site was accomplished by removing the negatively cha rged aspartate residue, which normally participates in a conflicting intera ction with the G4 nucleotide of the lambda B' site. We showed that, althoug h our mutants retain the ability to recombine their cognate att sites, they are unable to recombine lambda att sites.