Excision of oxidative cytosine modifications from gamma-irradiated DNA by Escherichia coli endonuclease III and human whole-cell extracts

The possible release from gamma-irradiated DNA of eight oxidatively modified cytosine bases by Escherichia coli endonuclease III was examined by trimethylsilylation and gas chromatography/electron impact/mass spectrometry. The results indicated that endonuclease III induced the release of 5-hydroxyhydantoin (1), 5-hydroxyuracil (2), cis-uracil 5,6-glycol (3), 5-hydroxycytosine (4), trans-uracil 5,6-glycol (5), and trans-1-carbamoyl-2-oxo-4,5-dihydroxyimidazolidine (8), The release of these products increased with the initial amount of damage in DNA, i,e., the dose of gamma-radiation (0-100 Gy), giving 4.6 +/- 1.0 fmol of 1,5.8 +/- 0.3 fmol of 2, 4.9 +/- 0.5 fmol of 3, 11.2 +/- 1.2 fmol of 4, 10.7 +/- 2.1 fmol of 5, and 1.5 +/- 0.5 fmol of 8, per microgram DNA per 10 Gy. In addition, we estimated that the relative rates of excision were 5 congruent to 3 > (1.2-fold) 1 > (1,5-fold) 4 > (3.3-fold) 2 on the basis of their initial yields in DNA and initial rates of release as a function of incubation time, The excision of 5-hydroxyuracil (2) and 5-hydroxycytosine (4) lesions was studied in greater detail by enzymatic digestion and HPLC coupled to electrochemical (EC) detection which determines the amounts of these products in DNA. The results showed that the excision of 4 was more efficient than that of 2 (2.7-fold) with greater than 50% of the lesions remaining in DNA after treatment. Finally, we examined the excision of products 2 and 4 from irradiated DNA (50 Gy) by whole human cell extracts, The release of product 2 into the hydrosylate was 5.2 +/- 1.4 fmol per microgram of DNA as measured by fluorobenzylation coupled to gas chromatography/electron capture negative-ion chemical ionization/mass spectrometry, In identical samples, the amount of product 2 was reduced by 45.0 +/- 2.6% (225 from 500 fmol per microgram of DNA) and that of product 4 by 7.0 +/- 3.1% (42 from 600 fmol per microgram of DNA) as measured by HPLC/EC analysis. (C) 1996 Academic Press, Inc.