title: Neocarzinostatin-induced hydrogen atom abstraction from C-4' and C-5' of the T residue at a d(GT) step in oligonucleotides: shuttling between deoxyribose attack sites based on isotope selection effects.
abstract: The thiol-activated neocarzinostatin chromophore cleaves duplex oligonucleotides containing the sequence-TGTTTGA-, producing 3'-phosphoglycolate and 3'-phosphate fragments at T, indicating the involvement of 4'- as well as 5'-chemistry at this residue. Substitution of deuterium for hydrogen at the C-4' position of the affected T leads to a kinetic isotope effect (kH/kD) of 4.0 on the formation of the glycolate-ended product, whereas deuterium at C-5' of the same T reveals kH/kD of 1.6 in the formation of the phosphate-ended product. The proportion of the products representing 4'- and 5'-chemistry can be shifted on the basis of isotope selection effects. A second product resulting from 4'-chemistry, the abasic site associated with 4'-hydroxylation, has been identified as an alkali-labile site, and as a pyridazine derivative formed after cleavage by hydrazine. A comparable isotope effect on its production (kH/kD = 3.7) relative to that of 3'-phosphoglycolate production is consistent with a common intermediate, a putative 4'-peroxy radical, in their formation. The formation of both products of 4'-chemistry is oxygen-dependent, and the internal partitioning between them (3'-phosphate or 3'-phosphoglycolate) is influenced by thiols. Moreover, the nitroaromatic radiation sensitizer misonidazole can substitute for dioxygen, yielding 3'-phosphoglycolate and alkali-labile 3'-phosphate ends, indicative of 4'-chemistry. In addition to the internal partitioning of 4'-chemistry, thiols also affect the overall extent of cleavage (4' plus 5') and the relative partitioning between both sites of attack (4' or 5').
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: L S
authors.initials: LS
authors.lastname: Kappen
authors.forename: I H
authors.initials: IH
authors.lastname: Goldberg
authors.forename: B L
authors.initials: BL
authors.lastname: Frank
authors.forename: L
authors.initials: L
authors.lastname: Worth
authors.forename: D F
authors.initials: DF
authors.lastname: Christner
authors.forename: J W
authors.initials: JW
authors.lastname: Kozarich
authors.forename: J
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D000255:Adenosine Triphosphate; D001483:Base Sequence; D003855:Deoxyribose; D003903:Deuterium; D006859:Hydrogen; D006860:Hydrogen Bonding; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D010761:Phosphorus Radioisotopes; D011865:Radioisotope Dilution Technique; D009353:Zinostatin
chemical_list: D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D003855:Deoxyribose; D006859:Hydrogen; D000255:Adenosine Triphosphate; D009353:Zinostatin; D003903:Deuterium