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

The Integrases of bacteriophages lambda and HK022 promote recombinatio n between DNA molecules that carry attachment sites. The two integrase s are about 70 % identical in sequence and catalyze nearly identical r eactions, but recognize different sets of sites. To identify the amino acids that determine this difference in specificity, we selected muta nts of lambda integrase with increased ability to recombine HK022 site s. This selection yielded eleven different amino acid substitutions at eight different positions. Three of the positions belong to a larger set that were identified as important for the lambda/HK022 specificity difference by analysis of chimeric integrases. Substitution of the HK 022 for the corresponding lambda residue at each of these three positi ons increased recombination of HK022 sites, and one double substitutio n, N99D-E319R, increased recombination to nearly wild-type HK022 level s. Mutations at the other five positions changed residues that are ide ntical in the wild-type proteins or are at positions identified by chi mera analysis as unimportant for the lambda/HK022 specificity differen ce. All of the mutants isolated by selection for increased recombinati on of HK022 sites retained considerable ability to recombine lambda si tes. However, we found that substitution of HK022 for lambda residues at three additional positions, S282P, G283K, and R287X, specifically r educed recombination of lambda sites. These three substitutions when c ombined with N99D and E319R were sufficient to change the specificity of lambda to that of HK022 integrase. The first three substitutions ac t principally to prevent recombination of lambda sites, and the second two to remove a barrier to recombination of HK022 sites. We suggest t hat many natural alterations in the specificity of protein-DNA interac tions occur by multi-step changes that first relax and then restrict s pecificity (C) 1995 Academic Press Limited