sup 32 P-postlabeling detection of thymine glycols: evaluation of adduct recoveries after enhancement with affinity chromatography, nuclease P1, nuclease S1, and polynucleotide kinase

Journal Article · Mon Apr 01 04:00:00 EST 1991 · Cancer Communications; (USA)

OSTI ID:5545625

Reddy, M V; Bleicher, W T; Blackburn, G R ^[1]

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1. Mobil Oil Corp., Princeton, NJ (USA)

Thymine glycol (Tg) is a product of DNA damage by oxygen radicals generated by oxidative mutagens and carcinogens and ionizing radiation. The highly sensitive {sup 32}P-postlabeling assay was validated and optimized for the measurement of Tg generated in vitro by the reaction of dTp or calf thymus DNA with osmium tetroxide (OsO{sub 4}). Adduct detection was enhanced by purification of Tg adducts using phenylboronate affinity chromatography or by preferential dephosphorylation of unmodified 3'-nucleotides with nuclease P1, nuclease S1, or polynucleotide kinase; Tg nucleotides were found to be resistant to limited enzymatic 3'-dephosphorylation. Two adducts were seen with OsO{sub 4}-modified dTp, which may have been cis-Tg adducts, because they were retained on a phenylboronate column, and because OsO{sub 4} selectively forms cis-Tg adducts. With OsO{sub 4}-modified DNA, several adducts were detected, two major derivatives of which coincided chromatographically with those seen in OsO{sub 4}-modified dTp. The recoveries of major adducts were similar before and after enrichment by different methods, indicating that Tg adducts were resistant to enzymatic dephosphorylation. The efficacy of labeling of the two major Tg adducts by polynucleotide kinase was optimal at 60 microM ATP and higher, whereas it was about 3%, 50%, and 80% of the optimal rate at 2, 10, and 30 microM, respectively. This was in contrast to our previous finding that only 0.25 microM ATP was needed for optimal labeling of benzoquinone-DNA adducts.

• OSTI ID: 5545625
• Journal Information: Cancer Communications; (USA), Journal Name: Cancer Communications; (USA) Vol. 3:4; ISSN 0955-3541; ISSN CNCME
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ADDUCTS
ALCOHOLS
ATP
AUTORADIOGRAPHY
AZINES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DNA
DNA ADDUCTS
DOSE-RESPONSE RELATIONSHIPS
ENZYMES
ESTERASES
GLYCOLS
HETEROCYCLIC COMPOUNDS
HYDROLASES
HYDROXY COMPOUNDS
IN VITRO
ION EXCHANGE CHROMATOGRAPHY
ISOTOPE APPLICATIONS
ISOTOPES
LABELLING
LIGHT NUCLEI
NUCLEASES
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHODIESTERASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PYRIMIDINES
RADIOASSAY
RADIOISOTOPES
SEPARATION PROCESSES
THYMINE
TRACER TECHNIQUES
TRANSFERASES
URACILS