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

Abstract

The long-wavelength ultraviolet (lambda approximately 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 approximately 1:10). By treating azidoacridine-photomodified DNA with piperidine at 90 degrees C, additional strand scissions are observed in a complex sequence dependent manner with an overall preference for T greater than or equal to G greater than C much greater than 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.

Chemical cleavage of plasmid DNA by glutathione in the presence of Cu(II) ions. The Cu(II)-thiol system for DNA strand scission

“…The other commonly used chemical cleavage systems include Cu(I)-bis-(1,10-phenanthroline) (Spassky & Sigman, 1985;Chu & Orgel, 1985; Franqois et Veal & Rill, 1989), metal-porphyrin complexes (Lown & Joshua, 1982;Lown et al, 1986;Bernadou et al, 1989;Groves & Farrell, 1989) and rhodium complexes (Kirshenbaum et al, 1988). Photochemical cleaving systems for DNA have also been described (Doan et al, 1987;Jeppesen et al, 1988;Saito et al, 1989;Jeppesen & Nielsen, 1989;Benimetskaya et al, 1989). The metal-complex systems above all require activation to induce strand scission, frequently achieved by means of a reducing agent but also by oxidizing agents in some situations (Sagripanti & Kraemer, 1989;Yamamoto et al, 1989).…”

Photolytic cleavage of DNA by nitrobenzamido ligands linked to 9-aminoacridines gives DNA polymerase substrates in a wavelength-dependent reaction

“…(Hertzberg & Dervan, 1984; Tullius & Dombroski, 1985; Sigman, 1986; Barton, 1986; Stubbe & Kozarich, 1987; Goyne & Sigman, 1987;Tullius, 1989). In some cases photochemical activation of the cleavage reaction has also been exploited (see, e.g., Freifelder et al, 1961; Friedman & Brown, 1978; Subramanian & Meares, 1985; Fleisher et al, 1986; Blacker et al, 1986; Ward et al, 1986;Buchardt et al, 1987; Nielsen et al, 1988a-c;Jeppesen et al, 1988; Behr, 1989; Veal & Kill, 1988; Jeppesen & Nielsen, 1989a,b; Uchida et al, 1989).…”

High resolution footprinting ofEcoRI and distamycin with Rh(phi)2(bpy)3+, a new photofootprinting reagent

“…An advantage of this method is that the activation of the DNA cleavage reaction is controlled by light, eliminating the need for adding other chemicals to the protein solution. These techniques include ultraviolet footprinting (14), photofootprinting in the presence of uranyl salts (15), and that in the presence of psoralen or its analogs (16). Ultraviolet light photofootprinting has been applied in vivo as well as in vitro.…”

“…The second technique, using uranyl salts, shows excellent sequence neutrality but high concentrations of the uranyl salts are required, which may perturb the protein interactions with the DNA or the DNA structure itself. The cleavage pattern with psoralen shows sequence preferences (16). Owing to these difficulties, despite the inherent advantages of light activation, these photofootprinting reagents have not been widely applied.…”