phosphorus-radioisotopes and thymine-glycol

We have used a 32P-postlabelling assay to examine the activity of purified Esherichia coli endonuclease IV, human apurinic/apyrimidinic endonuclease I and human cell-free extracts towards irradiated DNA. The assay can detect thymine glycols, 3'-phosphoglycolate groups and at least one other major lesion that has yet to be fully characterized. It was observed that endonuclease IV removed the phosphoglycolates and the uncharacterized lesion(s) suggesting that the latter are abasic sites with modified deoxyribose residues. The purified human enzyme acted only on the phosphoglycolate residues. Cell-free extract, prepared from A549 lung carcinoma cells by sonication or treatment with toluene, efficiently removed the phosphoglycolate and unknown lesions, but was less reactive towards thymine glycols. The extract was completely inactivated by heating at 60 degrees C for 10 min. Removal of the unknown product and phosphoglycolate did not require magnesium, but 1 mM EDTA did inhibit release of the latter. The cell-free extract exhibited substantially more activity towards native than heat-denatured DNA. A comparison of extracts prepared from 4 cell lines displaying a range of radiosensitivities, including an ataxia telangiectasia cell line, showed that all contained similar levels of repair activity towards the detectable lesions.

Topics: Cell Extracts; Cell Survival; Deoxyribonuclease I; Deoxyribonuclease IV (Phage T4-Induced); DNA; DNA Damage; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA, Single-Stranded; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli Proteins; Gamma Rays; Glycolates; Humans; Lyases; Magnesium; Nucleic Acid Denaturation; Phosphorus Radioisotopes; Thymine; Tumor Cells, Cultured

Topics: Base Sequence; Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Chromatography, Liquid; Deoxyadenine Nucleotides; Deoxyribonuclease (Pyrimidine Dimer); DNA Damage; DNA Repair; Endodeoxyribonucleases; Indicators and Reagents; Molecular Sequence Data; Molecular Weight; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Restriction Mapping; Saccharomyces cerevisiae; Substrate Specificity; Thymine

A 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' 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'-end of each molecule, they can be readily phosphorylated by T4 polynucleotide kinase and [gamma-32P]ATP and analyzed by polyacrylamide gel electrophoresis and reverse-phase HPLC. We 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 (i) a comparison of the radiation-induced products with those produced by OsO4 and KMnO4 and (ii) incubation of irradiated DNA with Escherichia coli endonuclease III and exonuclease III before analysis by the postlabeling procedure. This was confirmed by comigration of the radiogenic products with chemically synthesized markers. G values of 0.0022 and 0.0105 mumol J-1 were obtained for thymine glycol and phosphoglycolate production, respectively. The identity of the 5'-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.

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Deoxyribonuclease I; DNA; DNA Damage; DNA Repair; Genetic Markers; Glycolates; Hydrolysis; Hydroxides; Hydroxyl Radical; Osmium Tetroxide; Phosphodiesterase I; Phosphoric Diester Hydrolases; Phosphorus Radioisotopes; Phosphorylation; Polynucleotide 5'-Hydroxyl-Kinase; Pyrimidine Dimers; T-Phages; Thymine; X-Rays

Thymine glycol (Tg) is a product of DNA damage by oxygen radicals generated by oxidative mutagens and carcinogens and ionizing radiation. The highly sensitive 32P-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 (OsO4). 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 OsO4-modified dTp, which may have been cis-Tg adducts, because they were retained on a phenylboronate column, and because OsO4 selectively forms cis-Tg adducts. With OsO4-modified DNA, several adducts were detected, two major derivatives of which coincided chromatographically with those seen in OsO4-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.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Autoradiography; Benzene; Chromatography, Affinity; Chromatography, Thin Layer; DNA; DNA Damage; Dose-Response Relationship, Drug; In Vitro Techniques; Osmium Tetroxide; Phosphorus Radioisotopes; Polynucleotide 5'-Hydroxyl-Kinase; Single-Strand Specific DNA and RNA Endonucleases; Thymine