Gamma delta transposase. Purification and analysis of its interaction with a transposon end.
gamma delta, a member of the Tn3 family of prokaryotic transposons, encodes a transposase that binds to the 35-base pair (bp) terminal inverted repeats (IRs) which define the transposing DNA segment. The gamma delta transposase has been overexpressed, identified by molecular weight determination and by immunoblotting, and purified to homogeneity. Production of soluble transposase required the presence of Mg2+ prior to cell lysis. Fractions from a Sephacryl S-300 column contained levels of IR-binding activity that parallel the concentration of transposase, indicating that transposase alone is sufficient for binding to the ends of gamma delta. Hydroxyl radical footprinting indicated that transposase binds to one face of the DNA helix. The protected region extends across the IR and up to 17 bp into the flanking DNA. Integration host factor (IHF), which binds adjacent to transposase, also protects one face of the DNA helix and is shifted about 70 degrees around the helical axis from the transposase protection. Analysis of transposase-DNA complexes by electrophoresis on nondenaturing gels indicated that three complexes, two within the gel and one trapped at the well, result from specific interactions with the IR. The complex in the well and one complex in the gel were analyzed by methylation interference experiments. The results indicate that transposase interacts with specific base pairs between positions 10 and 37 of the IR, a region encompassing three consecutive major and minor grooves. Methylated bases at the very end of the transposon (positions 1-9) and in the flanking DNA did not inhibit transposase binding. Thus, although transposase seems to be in intimate contact throughout the IR of gamma delta and 17 bp of flanking DNA, specific base pair recognition needed for binding appears to be determined by the inner three-quarters of the IR.
Wiater
L A
LA
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
Grindley
N D
ND
D001665:Binding Sites; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008745:Methylation; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D019895:Transposases
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D009713:Nucleotidyltransferases; D019895:Transposases