title: Double-stranded damage of DNA.RNA hybrids by neocarzinostatin chromophore: selective C-1' chemistry on the RNA strand.
abstract: Glutathione-activated neocarzinostatin chromophore generates bistranded lesions in the hybrid formed by yeast tRNA(phe) and DNA complementary to its 31-mer 3' terminus. To elucidate the chemistry of the RNA cleavage reaction and to show that the lesions are double-stranded (ds), a series of shorter oligoribonucleotides containing the target sequence r(AGAAUUC).(GAATTCT) (underlining indicates major attack site) was studied as substrates. In addition to cleavage at both U residues, major damage was produced in the form of an abasic site at the U residues. Evidence for abasic site formation on the RNA strand was obtained from sequencing-gel analysis and measurement of uracil base release. Initial evidence for the ds nature of the damage came from experiments in which 2'-O-methyluridine was substituted for uridine in the RNA at one or both of the target sites. The site containing the substitution was not a target for cleavage or abasic site formation, and the particular T residue, staggered two nucleotides in the 3' direction on the complementary DNA strand, was cleaved significantly less. These studies were valuable in identifying the DNA ds partner of the RNA attack site. Direct evidence for ds lesions came from analysis of the products from a hairpin oligonucleotide construct in which the RNA and DNA strands were linked by four T residues and contained an internal 32P label at the 3' end of the RNA strand. Substitution of deuterium for hydrogen at the C-1' position of the U residues led to a substantial isotope effect (k1H/k2H = 3) upon the formation of the RNA abasic lesion and the RNA cleavage products, providing conclusive evidence for selective 1' chemistry. On the other hand, cleavage at the T residues on the complementary DNA strand involved C-5' hydrogen abstraction, as was also true for the T residue in an oligodeoxynucleotide analogue of the RNA strand. Chemical mechanisms to account for the RNA cleavage and abasic site formation via C-1' hydrogen abstraction are proposed.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: X
authors.initials: X
authors.lastname: Zeng
authors.forename: Z
authors.initials: Z
authors.lastname: Xi
authors.forename: L S
authors.initials: LS
authors.lastname: Kappen
authors.forename: W
authors.initials: W
authors.lastname: Tan
authors.forename: I H
authors.initials: IH
authors.lastname: Goldberg
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000737:chemistry / Q000494:pharmacology*; D001483:Base Sequence; D004249:DNA Damage; D003903:Deuterium; D053281:Enediynes; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000187:drug effects*; D009843:Oligoribonucleotides / Q000737:chemistry; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000187:drug effects*; D017423:Sequence Analysis, RNA; D009353:Zinostatin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000903:Antibiotics, Antineoplastic; D053281:Enediynes; D009692:Nucleic Acid Heteroduplexes; D009843:Oligoribonucleotides; D012360:RNA, Transfer, Phe; C032372:neocarzinostatin chromophore; D009353:Zinostatin; D003903:Deuterium