Specific binding of the biradical analog of neocarzinostatin chromophore to bulged DNA: implications for thiol-independent cleavage.
The enediyne anticancer antibiotic neocarzinostatin chromophore generates a single, site-specific break at a bulge in DNA in a thiol-independent reaction, involving intramolecular drug activation under general base catalysis [Kappen, L. S., & Goldberg, I. H. (1993) Biochemistry 32, 13138-13145]. As part of an effort to elucidate the three-dimensional structure of the active complex formed between the labile drug and bulged DNA, we have studied the binding of stable drug products generated in the course of the cleavage reaction with oligodeoxynucleotides containing the bulged structure. By use of fluorescence quenching, we have found that one drug product, which is also formed in the absence of bulged DNA and most closely resembles the biradical intermediate in the cleavage reaction, specifically binds bulged DNA with a Kd in the low micromolar range and competitively inhibits the cleavage reaction. Other drug products, including one formed only in the presence of bulged DNA, fail to bind to the bulged DNA. Implications of these results for the proposed mechanism of bulge-specific cleavage and for the role of the DNA bulge in generating a unique drug product are discussed.
Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115.
C F
CF
Yang
A
A
Stassinopoulos
I H
IH
Goldberg
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D018736:DNA Adducts; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D009353:Zinostatin / Q000737:chemistry*
D018736:DNA Adducts; D004247:DNA; D009353:Zinostatin