CONCERTED DNA RECOGNITION AND NOVEL SITE-SPECIFIC ALKYLATION BY DUOCARMYCIN-A WITH DISTAMYCIN-A

Duocarmycin A, a novel antitumor antibiotic, has a reactive cyclopropane ring, which has been reported to alkylate adenine at the 3' end of sequences of three or more consecutive A or T in DNA [Boger, D. L., et al. (1990) J. Am. Chem. Soc. 112, 8961-8971]. In order to study the DNA recognition, the reaction of DNA with duocarmycin A was performed in the presence of DNA ligands. Distamycin A, berenil, Hoechst 33258, and 4',6-diamidino-2-phenylindole (DAPI), which are minor-groove binders with affinity to A.T-rich sequences, were used. DNA-sequencing experiments showed that treatment of DNA with duocarmycin A plus distamycin A caused alkylation of guanine residues in G.C-rich sequences, which are not alkylated by duocarmycin A alone. Guanine alkylation by duocarmycin A was not observed with berenil, Hoechst 33258, or DAPI. HPLC product analysis showed that duocarmycin A reacted with a double-helical DNA octamer d(CCCCGGGG)2 in the presence of distamycin A to produce duocarmycin A-guanine adduct, while duocarmycin A alone did not react with the octamer. Chromomycin A3, which binds as a Mg(II)-coordinated dimer to G.C-rich sequences in the minor groove, inhibited the guanine alkylation by duocarmycin A in the presence of distamycin A. A footprinting experiment showed that there is a distamycin A-binding site close to the alkylated guanine residue. These results suggest that two different molecules, duocarmycin A and distamycin A, cooperatively recognize DNA sequences including consecutive G.C base pairs resulting in alkylation at the novel guanine sites. The cooperative drug recognition can be designated as ''concerted DNA recognition''.