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.