The molecular basis of co-operative DNA binding between lambda integrase and excisionase.
Higher-order nucleoprotein complexes often stabilize catalytic proteins in appropriate conformations for optimal activity and contribute to regulation during reactions requiring association of proteins and DNA. Formation of such complexes, known as intasomes, is required for site-specific recombination catalysed by bacteriophage Lambda Integrase protein (Int). Int-catalysed recombination is regulated by a second bacteriophage-encoded protein, Excisionase (Xis), which both stimulates excision and inhibits integration. To exert its effect, Xis binds co-operatively with Int, thereby inducing and stabilizing a DNA bend that alters the intasome structures formed during recombination. A rare int mutant, int 2268 ts, was reported (Enquist, L.W. and Weisberg, R.A. (1984) Mol Gen Genet 195: 62-69) to be more defective for excision than integration. Here, we have determined that this mutant Int protein contains an E47K substitution, and that the resultant excision-specific defect is due, at least in part, to destabilized interactions between Int and Xis. Analysis of several engineered substitutions at Int position 47 showed that a negatively charged residue is required for co-operative DNA binding between Int and Xis, and suggest that the Int-E47 residue may contact Xis directly. Substitutions at Int position 47 also affect co-operative binding among Int proteins at arm-type DNA sites, and thereby reduce the efficiency of both integration and excision. Collectively, these results suggest that a single surface of the Int amino-terminal domain mediates two alternate types of co-operative binding interactions.
D001287:Attachment Sites, Microbiological; D010582:Bacteriophage lambda / Q000201:enzymology*; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D019426:Integrases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011485:Protein Binding; D011995:Recombination, Genetic; D014764:Viral Proteins; D014775:Virus Activation / Q000235:genetics; D016662:Virus Integration / Q000235:genetics
D004269:DNA, Bacterial; D004279:DNA, Viral; D014764:Viral Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C063911:excisionase