DNA binding by mutants of Tn21 resolvase with DNA recognition functions from Tn3 resolvase.
Substitution of amino acids within the section of Tn21 resolvase that corresponds to a helix-turn-helix structure, with the equivalent residues from Tn3 resolvase, yields proteins that retain the ability to mediate recombination between res sites from Tn21. These proteins had no recombinational activity on res sites from Tn3, even when the complete recognition helix had been exchanged. In this study, the binding of these mutants of Tn21 resolvase to DNA fragments containing res from either Tn21 or Tn3 was analysed by DNase I footprinting and by gel retardation. With DNA containing res from Tn21, the mutants bound to all three of the binding sites for resolvase (I, II, and III) but with a lower affinity than wild-type Tn21 resolvase. No complexes were detected between Tn3 resolvase and Tn21 DNA. With DNA containing res from Tn3, both the mutants and wild-type Tn21 resolvase bound to sites II and III, forming similar complexes to those with Tn3 resolvase: some of the mutants had higher affinities for these two sites on Tn3 DNA than on Tn21 DNA. In contrast, at site I in res from Tn3 (the location of the recombinational cross-over), the derivatives of Tn21 resolvase formed aberrant complexes whose structures differed radically from that with Tn3 resolvase. Alterations in the amino acid sequence of resolvase, within the helix-turn-helix region, therefore modulate the affinity of the protein for its target sequence in the DNA, but the specificity of resolvase for recombination at its cognate res sites is determined by the resultant organization of the DNA-protein complex.
Avila
P
P
Department of Biochemistry, University of Bristol, U.K.
Ackroyd
A J
AJ
Halford
S E
SE
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000201:enzymology; D016296:Mutagenesis; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic / Q000502:physiology; D019895:Transposases; D045564:Transposon Resolvases
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D009713:Nucleotidyltransferases; C473490:Tn21 resolvase; D019895:Transposases; D045564:Transposon Resolvases; D003850:Deoxyribonuclease I