Identifying determinants of recombination specificity: Construction and characterization of mutant bacteriophage integrases

László Dorgai, Ezra Yagil, Robert A. Weisberg*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

The Integrases of bacteriophages λ and HK022 promote recombination between DNA molecules that carry attachment sites. The two integrases are about 70% identical in sequence and catalyze nearly identical reactions, but recognize different sets of sites. To identify the amino acids that determine this difference in specificity, we selected mutants of λ integrase with increased ability to recombine HK022 sites. 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 λ/HK022 specificity difference by analysis of chimeric integrases. Substitution of the HK022 for the corresponding λ residue at each of these three positions increased recombination of HK022 sites, and one double substitution, N99D-E319R, increased recombination to nearly wild-type HK022 levels. Mutations at the other five positions changed residues that are identical in the wild-type proteins or are at positions identified by chimera analysis as unimportant for the λ/HK022 specificity difference. All of the mutants isolated by selection for increased recombination of HK022 sites retained considerable ability to recombine λ sites. However, we found that substitution of HK022 for λ residues at three additional positions, S282P, G283K, and R287K, specifically reduced recombination of λ sites. These three substitutions when combined with N99D and E319R were sufficient to change the specificity of λ to that of HK022 integrase. The first three substitutions act principally to prevent recombination of λ sites, and the second two to remove a barrier to recombination of HK022 sites. We suggest that many natural alterations in the specificity of protein-DNA interactions occur by multi-step changes that first relax and then restrict specificity.

Original languageEnglish
Pages (from-to)178-188
Number of pages11
JournalJournal of Molecular Biology
Volume252
Issue number2
DOIs
StatePublished - 15 Sep 1995

Keywords

  • Attachment sites
  • Integrase
  • Lambda
  • Recombination specificity

Fingerprint

Dive into the research topics of 'Identifying determinants of recombination specificity: Construction and characterization of mutant bacteriophage integrases'. Together they form a unique fingerprint.

Cite this