Using the DNA sequencing technique of Maxam and Gilbert, we show that the protein antibiotic, neocarzinostatin, cleaves double-stranded qX174 DNA restriction fragments almost exclusively at deoxythymidylic and deoxyadenylic acid residues in a reaction requiring 2-mercaptoethanol. Overall, deoxythymidylic acid residues are attacked much more frequently than are deoxyadenylic acid residues, although there is variability in the attack rate for both nucleotides at different locations in the DNA molecule. While all deoxythymidylic acid residues are sites of scission by neocarzinostatin, not all deoxyadenylic acid residues are cleavage sites. There appears to be no clear-cut nucleotide sequence specificity in determining cleavage frequency. Single-stranded DNA is a very poor substrate for neocarzinostatin-induced scission; with one single-stranded DNA fragment, cleavage occurs at a position that is not attacked in double-stranded DNA. The possible significance for its biological activity of a drug that can attack both members of a DNA base pair is discussed. There is much evidence pointing toDNA alan important target in the action of the antitumor protein antibiotic, neocarzinostatin (1, 2). Thus, low levels of neocarzinostatin selectively inhibit DNA synthesis in sensitive bacterial and mammalian cells (3-6), induce degradation of existing DNA in bacteria (7), and produce breaks in DNA in mammalian cell lines (5,6,(8)(9)(10)(11). A correlation exists between the ability of the drug to induce breakage of cellular DNA in HeLa cells and its inhibition of DNA replication.and cell growth (6, 11). In addition, cells treated with neocarzinostatin possess chromosomal abnormalities (12, 13) and are blocked in the G2 phase of the cell cycle (13,14). Strong support for the involvement of DNA in the action of neocarzinostatin also comes from experiments in which it has been found that the drug is a mutagen for Escherichia coil and that the recA system is implicated in both mutagenicity and cell killing (15). Further, neocarzinostatin induces unscheduled DNA synthesis in lymphocytes (16) and DNA repair synthesis in intact HeLa cells and isolated nuclei (unpublished data).In addition to these effects on DNA structure and function in vivo, neocarzinostatin causes single-strand breaks in helical DNA in vitro in a reaction that is markedly stimulated by a sulfhydryl-containing compound (8, 11). The breaks in the DNA are not simple phosphodiester nicks (17, 18), but consist of gaps that bear 3'-and 5 '-phosphoryl. termini (19) and from which thymine (18,20) and smaller amounts of adenine (18) have been released. We now report on the use of the DNA sequencing technique of Maxam and Gilbert (21) to provide additional information on the nucleotide and possible sequence specificity of neocarzinostatin in the reaction in vitro. (21). Strand Separation. The two double-stranded (ds) 32P-labeled fragments described above were separated into their component single strands by alkaline treatment and gel electrophoresis (21), and the e...