Neocarzinostatin-induced hydrogen atom abstraction from C-4 prime and C-5 prime of the T residue at a d(GT) step in oligonucleotides: Shuttling between deoxyribose attack sites based on isotope selection effects

• Published in: Biochemistry (Easton) 1991-02, Vol.30:8
• Main Authors: Kappen, L.S., Goldberg, I.H., Frank B.L., Worth, L. Jr, Christner, D.F., Kozarich, J.W., Stubbe, J.
• Format: Article
• Language: English
• Subjects: 550201 - Biochemistry- Tracer Techniques; ALDEHYDES; ANTI-INFECTIVE AGENTS; ANTIBIOTICS; ANTINEOPLASTIC DRUGS; BASIC BIOLOGICAL SCIENCES; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; BIOLOGICAL EFFECTS; CARBOHYDRATES; DAYS LIVING RADIOISOTOPES; DEOXYRIBOSE; DEUTERIUM COMPOUNDS; DNA SEQUENCING; DRUGS; HYDROGEN COMPOUNDS; ISOTOPE EFFECTS; ISOTOPES; LIGHT NUCLEI; MONOSACCHARIDES; NEOCARCINOSTATIN; NUCLEI; NUCLEIC ACIDS; ODD-ODD NUCLEI; OLIGONUCLEOTIDES; ORGANIC COMPOUNDS; ORGANIC SULFUR COMPOUNDS; PENTOSES; PHOSPHORUS 32; PHOSPHORUS ISOTOPES; RADIOISOTOPES; RADIOMIMETIC DRUGS; SACCHARIDES; STRUCTURAL CHEMICAL ANALYSIS; THIOLS
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00222a005

The thiol-activated neocarzinostatin chromophore cleaves duplex oligonucleotides containing the sequence -TG{und T}TTGA-, producing 3{prime}-phosphoglycolate and 3{prime}-phosphate fragments at {und T}, indicating the involvement of 4{prime}- as well as 5{prime}-chemistry at this residue. Substitution of deuterium for hydrogen at the C-4{prime} position of the affected {und T} leads to a kinetic isotope effect (k{sub H}/k{sub D}) of 4.0 on the formation of the glycolate-ended product, whereas deuterium at C-5{prime} of the same {und T} reveals k{sub H}/k{sub D} of 1.6 in the formation of the phosphate-ended product. The proportion of the products representing 4{prime}- and 5{prime}-chemistry can be shifted on the basis of isotope selection effects. A second product resulting from 4{prime}-chemistry, the abasic site associated with 4{prime}-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 (k{sub H}/k{sub D} = 3.7) relative to that of 3{prime}-phosphoglycolate production is consistent with a common intermediate, a putative 4{prime}-peroxy radical, in their formation. The formation of both products of 4{prime}-chemistry is oxygen-dependent, and the internal partitioning between them (3{prime}-phosphate or 3{prime}-phosphoglycolate) is influenced by thiols. Moreover, the nitroaromatic radiation sensitizer misonidazole can substitute for dioxygen, yielding 3{prime}-phosphoglycolate and alkali-labile 3{prime}-phosphate ends, indicative of 4{prime}-chemistry. In addition to the internal partitioning of 4{prime}-chemistry, thiols also affect the overall extent of cleavage (4{prime} plus 5{prime}) and the relative partitioning between both sites of attack (4{prime} or 5{prime}).