Identifying Determinants of Recombination Specificity: Construction and Characterization of Chimeric Bacteriophage Integrases

Abstract

Bacteriophage integrases are members of a family of structurally related enzymes that promote recombination between DNA molecules that carry specific sites. Phages λ and HK022 encode closely related integrases that recognize different sets of sequences within the core regions of their respective attachments sites. To locate the amino acid residues that determine this difference in specificity, we isolated recombinant phages that produce chimeric integrases and measured the ability of these chimeras to promote recombination of λ and HK022 sites in vivo. A chimera that is of λ origin except for one HK022 residue at position 99 and 12 HK022 residues located between positons 279 and 329 had wild-type HK022 specificity and activity for both integrative and excisive recombination. Chimeras containing certain subsets of these 13 residues had incomplete specificity. The region around position 99 is not well-conserved in other members of the integrase family, but the 279-329 segment includes residues that are highly conserved and believed to be directly involved in catalysis. Many chimeras were inactive in recombining either HK022 or λ sites. Selection for mutants that restored activity to these chimeras revealed sets of residues that are likely to interact with each other.