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.(ABSTRACT TRUNCATED AT 250 WORDS)
Sun
D
D
Drug Dynamics Institute, College of Pharmacy, University of Texas, Austin 78712.
Hurley
L H
LH
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001345:Autoradiography; D001483:Base Sequence; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000187:drug effects / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000187:drug effects; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D000080890:Duocarmycins; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D007211:Indoles; D007933:Leucomycins / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes; D010957:Plasmids; D013604:T-Phages / Q000201:enzymology; D013698:Templates, Genetic
D000903:Antibiotics, Antineoplastic; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D000080890:Duocarmycins; D007211:Indoles; D007933:Leucomycins; D009692:Nucleic Acid Heteroduplexes; C018782:CC 1065; D004259:DNA-Directed DNA Polymerase