Identifying determinants of recombination specificity: construction and characterization of chimeric bacteriophage integrases.
Bacteriophage integrases are members of a family of structurally related enzymes that promote recombination between DNA molecules that carry specific sites. Phages lambda and HK022 encode closely related integrases that recognize different sets of sequences within the core regions of their respective attachment 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 lambda and HK022 sites in vivo. A chimera that is of lambda origin except for one HK022 residue at position 99 and 12 HK022 residues located between positions 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 lambda sites. Selection for mutants that restored activity to these chimeras revealed sets of residues that are likely to interact with each other.
Yagil
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E
Section on Microbial Genetics, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA.
Dorgai
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L
Weisberg
R A
RA
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D005075:Biological Evolution; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics*; D019426:Integrases; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D011995:Recombination, Genetic; D016415:Sequence Alignment; D016662:Virus Integration / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases