Quantitative determination of the 5-(hydroxymethyl)uracil moiety in the DNA of gamma-irradiated cells

Abstract

5-(Hydroxymethyl)uracil (HMUra) is a chemically stable derivative of thymine formed through the action of ionizing radiation which we previously identified in the DNA of gamma-irradiated HeLa cells [Teebor, G. W., Frenkel, K., & Goldstein, M. S. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 318-321]. In this report, we determine whether HMUra can be used as a marker of exposure of DNA to ionizing radiation. Dose-response curves for its formation in [3H]thymidine-labeled DNA were constructed by exposing the DNA to increasing amounts of gamma-radiation and measuring the HMUra content. DNA was irradiated both in solution and in intact cells. HMUra was identified as the 2'-deoxyribonucleoside 5-(hydroxymethyl)-2'-deoxyuridine (HMdU) by subjecting the irradiated DNA to enzymatic digestion and analyzing the mixture of 2'-deoxyribonucleosides by high-pressure liquid chromatography. The identity of the radiogenically formed HMdU was confirmed by acetylation and the structure of the acetyl derivative obtained by mass and nuclear magnetic resonance spectroscopies. At two different DNA concentrations in solution, the same number of thymidine moieties were converted to HMdU, indicating that within this range of concentration the formation of HMdU was mediated through the indirect action of ionizing radiation. Equal amounts of HMdU were formed in single- and double-stranded DNA at each radiation dose, indicating that DNA conformation did not affect HMdU formation. Surprisingly, the G value (number of HMdU molecules formed/100 eV) was higher in irradiated cellular DNA than in DNA irradiated in solution.(ABSTRACT TRUNCATED AT 250 WORDS)