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Specific Binding of Transposase to Terminal Inverted Repeats of Transposable Element Tn3
Hitoshi Ichikawa, Kaoru Ikeda, William L. Wishart and Eiichi Ohtsubo
Proceedings of the National Academy of Sciences of the United States of America
Vol. 84, No. 23 (Dec. 1, 1987), pp. 8220-8224
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/30433
Page Count: 5
You can always find the topics here!Topics: Genetic transposition, DNA, Transposons, Gels, Plasmids, Cellulose nitrate, Heparin, pH, Precipitates, Replicon
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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.
Proceedings of the National Academy of Sciences of the United States of America © 1987 National Academy of Sciences