Effect of the (+)-CC-1065-(N3-adenine)DNA Adduct on in Vitro DNA Synthesis Mediated by Escherichia Coli DNA Polymerase

(+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have shown that the potent cytotoxic and antitumor activities of (+)-CC-1065 are due to the ability of this compound to covalently modify DNA. (+)-CC-1065 reacts with duplex DNA to form an N3-adenine DNA adduct which lies in the minor groove of the DNA helix overlapping with a 5-base-pair region. As a consequence of covalent modification with (+)-CC-1065, the DNA helix bends into the minor groove and also undergoes winding and stiffening [Lee, C.-S., Sun, D., Kizu, R., & Hurley, L. H. (1991) Chem. Res. Toxicol. 4, 203-213]. In the studies described here, in which we have constructed site-directed DNA adducts on single-stranded DNA templates, we have shown that (+)-CC-1065 and select synthetic analogues, which have different levels of cytotoxicity, all show strong blocks against progression of Klenow fragment, E. coli DNA polymerase, and T4 DNA polymerase. The inhibition of bypass of drug lesions by polymerase could be partially alleviated by increasing the concentration of dNTPs and, to a small extent, by increasing polymerase levels. Klenow fragment binds equally well to a DNA template adjacent to a drug modification site and to unmodified DNA. These results taken together lead us to suspect that it is primarily inhibition of base pairing around the drug modification site and not prevention of polymerase binding that leads to blockage of DNA synthesis. Unexpectedly, the exact termination site of the in vitro DNA synthesis by Klenow fragment is not dependent on the species of covalently bound drug molecule but on the sequence to the 5' side of the drug-modified adenine. Misincorporation of dA for dG by Klenow fragment occurred at the secondary pausing site specifically for (+)-CC-1065 contained within the covalently modified sequence 5'-GATTA-3'. Although (+)-CC-1065 and its analogues evaluated in this study did not produce dramatically different effects on DNA polymerases when the drugs were bound to a single-stranded template, polymerization from a primer site containing a drug lesion in the duplex region did show a selective inhibitory effect with (+)-CC-1065 and (+)-AB'C'. When this observation is considered alongside results of experiments showing selective inhibition by these same compounds of T4 ligase and helicase II, the winding phenomena uniquely found with these compounds may be associated with the potent biological effect known as delayed lethality.