Site-specific cleavage at a DNA bulge by neocarzinostatin chromophore via a novel mechanism.
The chromophore of the anticancer drug neocarzinostatin (NCS-Chrom) oxidatively cleaves single-stranded or duplex DNA site-specifically in the absence of activating thiol provided that the DNA contains a bulged structure. Point mutations, deletions, and insertions in the DNA analogue and its complement of the 3'-terminus of yeast tRNA(Phe) show that for a single-stranded DNA to be cleaved by NCS-Chrom the DNA must generate a hairpin structure with an apical loop and at least a two-base-pair stem hinged to a region of duplex structure via a bulge containing a target nucleotide at its 3' side. The size of the loop is not critical so long as it contains at least three nucleotides; the bulge requires a minimum of two nucleotides but must have fewer than five. With a notable exception involving base-pair changes immediately 3' to the bulge, base changes in the bulge and base-pair changes immediately 5' to the bulge retain substrate activity for NCS-Chrom. Maintenance of the bulged structure requires stable duplex regions on each side of the bulge. A similar bulged structure, but lacking a loop, formed by the annealing of a linear 8-mer and a 6-mer is an excellent target for cleavage in the thiol-independent reaction. Drugs such as netropsin, which sequester the DNA into nonbulge containing structures inhibit the reaction. In the absence of O2 strand cleavage is blocked and quantitatively replaced by a presumed drug-DNA covalent adduct.(ABSTRACT TRUNCATED AT 250 WORDS)
Kappen
L S
LS
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Goldberg
I H
IH
D000693:Anaerobiosis; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009429:Netropsin / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000737:chemistry; D009353:Zinostatin / Q000737:chemistry*
D009838:Oligodeoxyribonucleotides; D013438:Sulfhydryl Compounds; D009429:Netropsin; D004247:DNA; D009353:Zinostatin