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32P-postlabeling detection of radiation-induced DNA damage: identification and estimation of thymine glycols and
1Radiobiology Program, Cross Cancer Institute, Edmonton, Alberta, Canada.
Abstract:
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.
Insights
A new 32P-postlabeling assay detects DNA damage at the femtomole level. This method identifies radiation-induced thymine glycols and phosphoglycolate groups, implicating hydroxyl radicals in DNA lesion formation.
Area of Science:
- Molecular Biology
- Radiation Chemistry
- Biochemistry
Background:
- DNA damage is a critical factor in radiation-induced cellular effects.
- Accurate quantification of DNA lesions is essential for understanding radiation biology.
- Existing methods for detecting DNA damage have limitations in sensitivity and specificity.
Purpose of the Study:
- To develop a sensitive assay for detecting and quantifying radiogenic DNA base and sugar lesions.
- To elucidate the role of hydroxyl radicals in DNA damage formation.
- To characterize specific DNA lesions induced by ionizing radiation.
Main Methods:
- Development of a 32P-postlabeling assay for DNA lesion detection.
- Enzymatic digestion of irradiated DNA with nucleases and alkaline phosphatase.
- Phosphorylation of lesion-bearing dinucleoside monophosphates using T4 polynucleotide kinase and [gamma-32P]ATP.
- Analysis of labeled products by polyacrylamide gel electrophoresis and reverse-phase HPLC.
- Identification of lesions using chemical treatments, enzymatic assays (Endonuclease III, Exonuclease III, Nuclease P1), and comparison with synthesized markers.
Main Results:
- The 32P-postlabeling assay detects DNA damage at the femtomole level.
- A linear induction of total DNA damage was observed with radiation doses from 5-50 Gy.
- Damage induction was significantly reduced in the presence of 1 M DMSO, suggesting hydroxyl radical involvement.
- Thymine glycols and phosphoglycolate groups were identified as major radiation-induced lesions.
- G values for thymine glycol and phosphoglycolate production were determined to be 0.0022 and 0.0105 mumol J-1, respectively.
- Analysis of nearest-neighbor bases indicated a nonrandom interaction between hydroxyl radicals and DNA.
Conclusions:
- The developed 32P-postlabeling assay is highly sensitive for quantifying radiogenic DNA lesions.
- Hydroxyl radicals play a significant role in the formation of DNA base and sugar damage following irradiation.
- The characterization of thymine glycols and phosphoglycolates provides insights into the mechanisms of radiation-induced DNA damage.