Abstract
The pseudo EcoRI site GAATTA in the U3 region of the long terminal repeat of human immunodeficiency virus, which is flanked by a 26-base pair oligopurine tract, is readily nicked by either EcoRI or RsrI. The strand-specific nick occurs predominantly between the G and A residues and is independent of negative supercoiling. Other GAATTA sites surrounded by random (non-oligopurine) sequences are not nicked by these restriction endonucleases. However, other types and lengths of oligopurine tracts are effective in inducing the nicking in neighboring GAATTA sites. Hence, we propose that the flanking oligopurine tracts induce an altered DNA conformation on the GAATTA target site which may be similar to the transition state induced by EcoRI when binding to its canonical recognition site. Gel retardation analyses on restriction fragments containing the oligopurine-GAATTA-oligopurine sequences suggest the presence of helical axis distortions which are consistent with this interpretation.
Highlights
A DNA Conformational Alteration Induced by a Neighboring Oligopurine Tract on GAATTA Enables Nicking by EcoRI*
Other GAATTA sites surrounded by random sequences are not nicked by these restriction endonucleases
Other types and lengths of oligopurine tracts are effective ininducing the nicking in neighboring GAATTA sites
Summary
0 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 266, No 25, Issue of September 5, pp. 16786-16’790,1991 Printed in U.S.A. 16786-16’790,1991 Printed in U.S.A. A DNA Conformational Alteration Induced by a Neighboring Oligopurine Tract on GAATTA Enables Nicking by EcoRI*. Other GAATTA sites surrounded by random (non-oligopurine) sequences are not nicked by these restriction endonucleases. Other types and lengths of oligopurine tracts are effective ininducing the nicking in neighboring GAATTA sites. We propose that the flanking oligopurine tracts induce an altered DNA conformation on the GAATTA target site which may besimilar to the transition state induced by that sequence was nicked by EcoRI under conditions that normally would not allow cleavageor nicking. Systematic studies on mutated EcoRI sites (26, taining the oligopurine-GAATTA-oligopurine se- 29) revealed that several kinds of changes in the recognition quences suggest the presence of helical axis distortions sequence can greatly increase the energetic barrier for the which are consistent with thisinterpretation.
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