Footprinting with MPE.Fe(II). Complementary-strand analyses of distamycin- and actinomycin-binding sites on heterogeneous DNA.

We recently reported a direct technique for determining the binding sites of small molecules on naturally occurring heterogeneous DNA (Van Dyke et al. 1982). Methidiumpropyl-EDTA.Fe(lI) (MPE.Fe[I~) (Hertzberg and Dervan 1982) cleaves double-helical DNA with low sequence-specificity (Van Dyke et al. 1982). Using a combination of MPE-Fe(II) partial cleavage of drugprotected DNA fragments and Maxam-Gilbert sequencing methods, we determined the drug-protected sites on one strand of a double-helical fragment from pBR322 for the intercalator actinomycin D (Goldberg et al. 1962; Muller and Crothers 1968; Wells and Larson 1970; Sobell 1973; Krugh 1981; Patel et al. 1981; Takusagawa et al. 1982) and the minor-groove binders netropsin and distamycin A (Luck et al. 1974; Wartell et al. 1974; Zimmer 1975; Berman et al. 1979; Krylov et al. 1979). Netropsin and distamycin A gave identical DNA-cleavage inhibition patterns or footprints in regions rich in dA.dT base pairs. Actinomycin D afforded a completely different footprint pattern centered around one or more dG.dC base pairs (Van Dyke et al. 1982). (See Fig. 1 for structures of MPE.Fe[II], actinomycin D, and distafiaycin A.) We are not certain whether there is a one-to-one correspondence between a protected region on one strand of the double-helical DNA fragment and the drugbinding site on the DNA. We presume that the reaction responsible for DNA strand scission by MPE.Fe(II) is an oxidative cleavage of the deoxyribose ring analogous to mechanisms postulated for bleomycin activity (Hecht 1979; Burger et al. 1981). The different accessibility of adjacent deoxyribose rings to the propyl-EDTA.Fe(II) moiety in the unprotected site bound by MPE might result in asymmetric footprints on opposite DNA strands. From model building with right-handed, double-helical DNA and MPE.Fe(II), we expect the footprint on each DNA strand to be shifted at least 1 bp to the 3' side of the drug-binding site (Fig. 2). Greater precision in identifying the boundaries of the preferred drug-binding sites would be afforded by an analysis of opposite strands. We report here complementary-strand analyses of distamycin and actinomycin binding on heterogeneous DNA using the MPE-Fe(II) footprinting technique. Two DNA restriction fragments, 117 bp and 168 bp in length, each containing regions of identical sequence, were prepared with 3' -end labeling on complementary strands. DNA-cleavage inhibition patterns resulting from MPE.Fe(II) cleavage of the drug-protected DNA restriction fragments provide the opposite-strand footprints (Fig. 3). This affords a more precise location of the preferred binding sites on 50 bp of heterogeneous DNA for distamycin and actinomycin.