Interactions of the integrase protein of the conjugative transposon Tn916 with its specific DNA binding sites.
The binding of two chimeric proteins, consisting of the N-terminal or C-terminal DNA binding domain of Tn916 Int fused to maltose binding protein, to specific oligonucleotide substrates was analyzed by gel mobility shift assay. The chimeric protein with the N-terminal domain formed two complexes of different electrophoretic mobilities. The faster-moving complex, whose formation displayed no cooperativity, contained two protein monomers bound to a single DNA molecule. The slower-moving complex, whose formation involved cooperative binding (Hill coefficient > 1.0), contained four protein monomers bound to a single DNA molecule. Methylation interference experiments coupled with the analysis of protein binding to mutant oligonucleotide substrates showed that formation of the faster-moving complex containing two protein monomers required the presence of two 11-bp direct repeats (called DR2) in direct orientation. Formation of the slower-moving complex required only a single DR2 repeat. Binding of the N-terminal domains in vivo could serve to position two Int monomers on the DNA near each end of the transposon and assist in bringing together the ends of the transposon so that excision can occur. The chimeric protein with the C-terminal domain of Int also formed two complexes of different electrophoretic mobilities. The major, slower-moving complex, whose formation involved cooperative binding, contained two protein molecules bound to one DNA molecule. This finding suggested that while the C-terminal domain of Int can bind DNA as a monomer, a cooperative interaction between two monomers of the C-terminal domain may help to bring the ends of the transposon together during excision.
Jia
Y
Y
Department of Microbiology and Immunology, Emory University, Atlanta, Georgia 30322, USA.
Churchward
G
G
D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003227:Conjugation, Genetic; D016172:DNA Fingerprinting; D019175:DNA Methylation; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic
D002352:Carrier Proteins; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D019426:Integrases