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Nucleic Acids Research, 1988, Vol. 16, No. 13 5755-5770
© 1988

Articles

Photocleavage of DNA and photofootprinting of E.coli RNA polymerase bound to promoter DNA by azido-9-acridinylamines

Claus Jeppesen, Ole Buchardt1, Ulla Henriksen1 and Peter E. Nielsen*

Department of Biochemistry B, The Panum Institute, University of Copenhagen Blegdamsvej 3, DK-2200 Copenhagen N 1The Bioorganic Group, Chemical Laboratory II, The H.C. Ørsted Institute, University of Copenhagen Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark

*To whom correspondence should be addressed

Received April 29, 1988. Revised June 1, 1988. Accepted June 1, 1988.

The long-wavelength ultraviolet ({lambda} {small tilde} 420 nm) radiation induced reaction between 6-azido-2-methoxy-9-acridinylamines and supercoiled plasmid DNA results in single strand scissions and formation of covalent adducts (ratio {small tilde} 1:10). By treating azidoacridine-photomodified DNA with piperidine at 90° C, additional strand scissions are observed in a complex sequence dependent manner with an overall preference for T ≥ G > C >> A. The resulting DNA fragments migrate as 5'-phosphates in polyacrylamide gels.

Photofootprinting of the binding site of RNA-polymerase on promoter DNA is demonstrated with an azido-9-acridinylamino-octamethylene-9-aminoacridine. Similar experiments using 9-amino-6-azido-2-methoxyacridine indicate that this reagent recognizes changes in the DNA conformation induced by RNA polymerase binding, in relation to open complex formation.


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