IDENTIFYING DETERMINANTS OF RECOMBINATION SPECIFICITY - CONSTRUCTION AND CHARACTERIZATION OF CHIMERIC BACTERIOPHAGE INTEGRASES

Bacteriophage integrases are members of a family of structurally relat ed enzymes that promote recombination between DNA molecules that carry specific sites. Phages lambda and HK022 encode closely related integr ases that recognize different sets of sequences within the core region s of their respective attachment sites. To locate the amino acid resid ues that determine this difference in specificity, we isolated recombi nant phages that produce chimeric integrases and measured the ability of these chimeras to promote recombination of lambda and HK022 sites i n vivo. A chimera that is of lambda origin except for one HK022 residu e at position 99 and 12 HK022 residues located between positions 279 a nd 329 had wild-type HK022 specificity and activity for both integrati ve and excisive recombination. Chimeras containing certain subsets of these 13 residues had incomplete specificity The region around positio n 99 is not well-conserved in other members of the integrase family, b ut the 279-329 segment includes residues that are highly conserved and believed to be directly involved in catalysis. Many chimeras were ina ctive in recombining either HK022 or lambda sites. Selection for mutan ts that restored activity to these chimeras revealed sets of residues that are likely to interact with each other. (C) 1995 Academic Press L imited