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 λ catalyse similar site-specific integration and excision reactions between specific DNA regions known as attachment (att) sites. However, the Int proteins of HK022 and λ are unable to catalyse recombination between non-cognate att sites. The att sites of both phages contain weak binding sites for Int, known as 'core-type' sites. Negatively acting nucleotide determinants associated with specific core sites (λ B', HK022 B', HK022 a) are responsible for the barrier to non-cognate recombination. In this study, we used challenge phages to demonstrate that the λ 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 λ B' core site. Two mutants, D99N and D99A, have changed a residue in the core-binding (CB) domain, which may be directly contacting the core site DNA. We suggest that binding to the λ B' site was accomplished by removing the negatively charged aspartate residue, which normally participates in a conflicting interaction with the G4 nucleotide of the λ B' site. We showed that, although our mutants retain the ability to recombine their cognate att sites, they are unable to recombine λ att sites.