Specific binding of transposase to terminal inverted repeats of transposable element Tn3.
Tn3 transposase, which is required for transposition of Tn3, has been purified by a low-ionic-strength-precipitation method. Using a nitrocellulose filter binding assay, we have shown that transposase binds to any restriction fragment. However, binding of the transposase to specific fragments containing the terminal inverted repeat sequences of Tn3 can be demonstrated by treatment of transposase-DNA complexes with heparin, which effectively removes the transposase bound to the other nonspecific fragments at pH 5-6. DNase I "footprinting" analysis showed that the transposase protects an inner 25-base-pair region of the 38-base-pair terminal inverted repeat sequence of Tn3. This protection is not dependent on pH. Interestingly, binding of the transposase to the inverted repeat sequences facilitates DNase I to nick at the end of the Tn3 sequence. It was also observed that the transposase protects the end regions of restriction fragments with a cohesive sequence at their 5' end or with a flush end from DNase I cleavage. The specific and nonspecific binding of transposase to DNA is ATP-independent.
Ichikawa
H
H
Institute of Applied Microbiology, University of Tokyo, Japan.
Ikeda
K
K
Wishart
W L
WL
Ohtsubo
E
E
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D004262:DNA Restriction Enzymes; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics; D009713:Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D019895:Transposases
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; D019895:Transposases; D004262:DNA Restriction Enzymes; D003850:Deoxyribonuclease I