Use of a postlabelling assay to examine the removal of radiation-induced DNA lesions by purified enzymes and human cell extracts.
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.
Weinfeld
M
M
Department of Oncology, University of Alberta, Cross Cancer Institute, Edmonton, Canada. mweinfel@gpu.srv.ualberta.ca
Lee
J
J
Ruiqi
G
G
Karimi-Busheri
F
F
Chen
D
D
Allalunis-Turner
J
J
D002457:Cell Extracts; D002470:Cell Survival; D004247:DNA / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D003850:Deoxyribonuclease I / Q000378:metabolism*; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005720:Gamma Rays / Q000009:adverse effects; D006016:Glycolates / Q000378:metabolism; D006801:Humans; D008190:Lyases / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D009691:Nucleic Acid Denaturation; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D013941:Thymine / Q000031:analogs & derivatives / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002457:Cell Extracts; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D006016:Glycolates; D010761:Phosphorus Radioisotopes; C029389:thymine glycol; D004247:DNA; D003850:Deoxyribonuclease I; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; C013193:phosphoglycolate; D008274:Magnesium; D013941:Thymine