Characterization of a novel covalent monoadduct on the RNA overhang of an RNA-DNA hybrid induced by antitumor antibiotic neocarzinostatin.
Neocarzinostatin chromophore (NCS-chrom) has been found previously to induce thiol-dependent abasic site formation and cleavage on the RNA strand of RNA-DNA hybrids. When a four-base nucleotide was deleted from the 5' end of the DNA strand, however, an unexpected product with slow mobility, potentially a drug adduct or interstrand cross-linked material, was observed instead. End-labeling of the RNA and DNA strands showed that only the RNA strand is present in the product. The product, isolated from a denaturing polyacrylamide gel, has a fluorescence spectrum similar to that of activated NCS-chrom, and thiols with different charges give rise to products with various mobilities on the gel, indicating that the product consists of a drug adduct with NCS-chrom and thiol attached to the RNA strand of the hybrid. Mass spectroscopic (MS) analysis shows that it is a monoadduct. Chemical sequencing of 5'- and 3'-end-labeled monoadduct and MS/MS data show that drug is attached only to the U9 of the RNA in the hybrid 5'-r(CACAGAAUU9CG)/5'-d(TTCTGTG). Unlike most other DNA adducts and cross-linked products found previously, this adduct can be formed readily in the presence of atmospheric oxygen. 2'-O-Methyl and 2'-H substitutions and nucleotide replacements on the 4-base RNA overhang affect monoadduct formation to different extents, reflecting the existence of a tight three-dimensional binding pocket for the activated drug. Stability of drug adduct to heat and aniline-HOAc treatment suggest that the drug is covalently bound to the ribose of U9. Since no deuterium isotope selection effect was observed for C-1' of U9, it is possible, although not required, that the drug abstracts H from another hydrogen source on the sugar of the targeted ribonucleotide.
Zheng
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Liu
C L
CL
Xi
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Z
Smith
R D
RD
Goldberg
I H
IH
D000332:Aerobiosis; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001482:Base Composition; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000737:chemistry* / Q000187:drug effects; D018736:DNA Adducts / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D004249:DNA Damage; D053281:Enediynes; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D010100:Oxygen; D012313:RNA / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014529:Uridine / Q000031:analogs & derivatives / Q000235:genetics; D009353:Zinostatin / Q000031:analogs & derivatives / Q000494:pharmacology
D000903:Antibiotics, Antineoplastic; D018736:DNA Adducts; D053281:Enediynes; D013438:Sulfhydryl Compounds; C052202:2'-O-methyluridine; D012313:RNA; C032372:neocarzinostatin chromophore; D004247:DNA; D009353:Zinostatin; D010100:Oxygen; D014529:Uridine