Complementation of bacteriophage lambda integrase mutants: evidence for an intersubunit active site.
Site-specific recombination of bacteriophage lambda starts with the formation of higher-order protein--DNA complexes, called 'intasomes', and is followed by a series of steps, including the initial DNA cleavage, top-strand exchange, branch migration and bottom-strand exchange, to produce recombinant products. One of the intasomes formed during excisive recombination (the attL complex) is composed of the phage-encoded integrase (Int), integration host factor (IHF) and one of the recombination substrates, attL DNA. Int is the catalytic recombinase and has two different DNA binding domains. When IHF is present, Int binds to two types of sites in attL DNA, the three arm-type sites (P'123) and the core-type sites (B and C') where the reciprocal strand exchange takes place. The Tyr342 residue of Int serves as a nucleophile during strand cleavage and covalently attaches to the DNA through a phosphotyrosyl bond. In vitro complementation assays have been performed for strand cleavage using attL suicide substrates and mutant proteins containing amino acid substitutions at residues conserved in the integrase family of recombinases. We demonstrate that at least two Int monomers are required to form the catalytically-competent species that performs cleavage at the B site. It is likely that the active site is formed by two Int monomers.
Han
Y W
YW
Department of Microbiology, College of Medicine, University of Illinois at Urbana-Champaign 61801.
Gumport
R I
RI
Gardner
J F
JF
D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D005816:Genetic Complementation Test; D019426:Integrases; D007700:Kinetics; D016296:Mutagenesis; D009154:Mutation; D013379:Substrate Specificity
D004279:DNA, Viral; D004254:DNA Nucleotidyltransferases; D019426:Integrases