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The use of isotopic labels to probe the mechanism of DNA oxidation by iron bleomycin

Thesis/Dissertation ·
OSTI ID:6414806
When the antitumor antibiotic bleomycin is activated anaerobically with Fe(III) and hydrogen peroxide or with Fe(II) and limiting oxygen, the DNA products are free nucleic acid base and an oxidatively damaged sugar lesion which undergoes strand scission when treated with alkali. Stabilization of the initial product by borohydride reduction and digestion by P{sub 1} nuclease and alkaline phosphatase afforded 2{double prime}-deoxypentitol-3{double prime}-O-5{prime}-phospho-2{prime}-deoxypurine nucleosides that accounted for 99, 81 and 48% of the pyrimidine base released from d(CGCGCG), poly(dA-dU) and poly(dG-dC), respectively. Further enzymatic degradation yielded 2-deoxy-D-erythro-pentitol and 2-deoxy-L-threo-pentitol which were identified by mass spectrometry. The 2{prime}-deoxypentos-4{prime}-ulose product arising from the interaction of d(CGCGCG) with bleomycin was 86 and 97% {sup 18}O-labeled at C-4{prime} at pH 9.0 and 7.8, respectively, when limiting {sup 16}O-labeled oxygen was used to activate bleomycin in {sup 18}O-water. Either complete isotopic exchange between solvent and a high-valent iron-oxo species of bleomycin or the equivalent of a 1e{sup {minus}} oxidation of the presumed 4 carbon-centered radical of DNA are required to account for these findings. When oxygen is supplied in excess of what is required to activate bleomycin, the C3{prime}-C4{prime} bond of DNA is ruptured to yield transbase propenals and oligonucleotides bearing 5-phosphate and 3-phosphoglycolate termini. Kinetics study of base propenal formation from a DNA-bound precursor and release of {sup 3}H from pro R and pro S poly(dA-(2-{sup 3}H)dU) showed that reported rapid release compared penal formation was the result of specific release from the pro R position and was not a consequence of the base release pathway nor nonstereospecific enolization of 2{prime} hydrogens.
Research Organization:
Wisconsin Univ., Madison, WI (USA)
OSTI ID:
6414806
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKALINE PHOSPHATASE
ANTI-INFECTIVE AGENTS
ANTIBIOTICS
ANTIMITOTIC DRUGS
ANTINEOPLASTIC DRUGS
BIOLOGICAL EFFECTS
BLEOMYCIN
BOROHYDRIDES
BORON COMPOUNDS
DNA
DRUGS
ELEMENTS
ENZYMES
ESTERASES
EVEN-EVEN NUCLEI
HYDROGEN COMPOUNDS
HYDROLASES
IRON
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MASS SPECTROSCOPY
METABOLISM
METALS
NUCLEASES
NUCLEI
NUCLEIC ACIDS
ORGANIC COMPOUNDS
OXYGEN 16
OXYGEN 18
OXYGEN ISOTOPES
PHOSPHATASES
PHOSPHODIESTERASES
SPECTROSCOPY
STABLE ISOTOPES
TRACER TECHNIQUES
TRANSITION ELEMENTS
TRITIUM COMPOUNDS