Identifying determinants of recombination specificity: construction and characterization of mutant bacteriophage integrases.
The Integrases of bacteriophages lambda 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 lambda 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 lambda/HK022 specificity difference by analysis of chimeric integrases. Substitution of the HK022 for the corresponding lambda 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 lambda/HK022 specificity difference. All of the mutants isolated by selection for increased recombination of HK022 sites retained considerable ability to recombine lambda sites. However, we found that substitution of HK022 for lambda residues at three additional positions, S282P, G283K, and R287K, specifically reduced recombination of lambda sites. These three substitutions when combined with N99D and E319R were sufficient to change the specificity of lambda to that of HK022 integrase. The first three substitutions act principally to prevent recombination of lambda 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.
Dorgai
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L
Section on Microbial Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Yagil
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E
Weisberg
R A
RA
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics*; D019426:Integrases; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D016415:Sequence Alignment; D016662:Virus Integration / Q000235:genetics*
D003062:Codon; D011993:Recombinant Fusion Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases