sup 32 P-postlabeling detection of radiation-induced DNA damage: Identification and estimation of thymine glycols and phosphoglycolate termini

Journal Article · Tue Jan 29 04:00:00 EST 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00218a031· OSTI ID:5510474

Weinfeld, M; Soderlind, K J.M. ^[1]

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1. Cross Cancer Institute, Edmonton, Alberta (Canada)

A {sup 32}P-postlabeling assay has been developed that permits detection of several radiogenic base and sugar lesions of DNA at the femtomole level. The technique is based on the inability of DNase I and snake venom phosphodiesterase to cleave the internucleotide phosphodiester bond immediately 5{prime} to the site of damage so that complete digestion of irradiated DNA with these nucleases and alkaline phosphatase yields lesion-bearing dinucleoside monophosphates. Because these fragments contain an unmodified nucleoside at the 5{prime}-end of each molecule, they can be readily phosphorylated by T4 polynucleotide kinase and ({gamma}-{sup 32}P)ATP and analyzed by polyacrylamide gel electrophoresis and reverse-phase HPLC. The authors observed a linear induction of total damage in DNA irradiated with 5-50 Gy. Virtually no damage was detected when the DNA was irradiated in solution containing 1 M DMSO, implicating hydroxyl radicals in the formation of these lesions. Evidence for the presence of thymine glycols and phosphoglycolate groups came from (1) a comparison of the radiation-induced products with those produced by OsO{sub 4} and KMnO{sub 4} and (2) incubation of irradiated DNA with Escherichia coli endonuclease 3 and exonuclease 3 before analysis by the postlabeling procedure. This confirmed by comigration of the radiogenic products with chemically synthesized markers. The identity of the 5{prime}-nucleotide of each isolated compound was obtained by nuclease P1 digestion. This analysis of nearest-neighbor bases to thymine glycols and phosphoglycolates indicated a nonrandom interaction between radiation-induced hydroxyl radicals and DNA.

• OSTI ID: 5510474
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:4; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ADDUCTS
ALCOHOLS
AZINES
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHALCOGENIDES
CHEMICAL REACTIONS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DNA
DNA ADDUCTS
DOSE-RESPONSE RELATIONSHIPS
ENZYMATIC HYDROLYSIS
ESCHERICHIA COLI
GLYCOLS
HETEROCYCLIC COMPOUNDS
HYDROLYSIS
HYDROXY COMPOUNDS
HYDROXYL RADICALS
INHIBITION
ISOTOPES
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
LYSIS
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OSMIUM COMPOUNDS
OSMIUM OXIDES
OXIDES
OXYGEN COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PYRIMIDINES
RADICALS
RADIOINDUCTION
RADIOISOTOPES
REFRACTORY METAL COMPOUNDS
SEPARATION PROCESSES
SOLVOLYSIS
THYMINE
TRANSITION ELEMENT COMPOUNDS
URACILS