Faculty Bibliography

A methodology for the separation of radiation-induced thymine derivatives in DNA using high pressure liquid chromatography is presented. DNA was subjected to enzymatic hydrolysis yielding 2'-deoxyribonucleosides and the hydrolysate cochromatographed with marker compounds. Confirmation of the presence of derivatives was accomplished by chromatography on Sephadex LH-20 and microderivatization. The method separates free bases from nucleosides allowing for identification of spontaneously released bases or those released through the action of repair enzymes. The results indicate that most of the thymine derivatives formed in irradiated cellular DNA were the same as those found in DNA irradiated in solution. However, the major cellular derivative was not present in the latter. This derivative was identified as 5-hydroxymethyl-2'-deoxyuridine (HMdU). HMdU has previously been shown to be cytotoxic to cells in culture and caused diarrhea and bone marrow failure when administered to mice. Thus, the presence of this radiation-induced thymine derivative in cellular DNA correlates with the known effects of ionizing radiation on cells and animals

5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) is formed in DNA by chemical oxidants and ionizing radiation. We describe the separation of thymine glycol, 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol), thymine, and thymidine by high-pressure liquid chromatography (HPLC). Enzymatic hydrolysates of chemically oxidized or gamma-irradiated single-stranded DNA were cochromatographed with 14C-containing marker compounds. In chemically oxidized DNA, thymidine glycol was the major derivative formed. In addition, there were four rapidly eluting thymine-derived components. In irradiated DNA, thymidine glycol constituted about 5% of the modified thymines, and the rapidly eluting fractions were proportionately increased. DNA isolated from gamma-irradiated and nonirradiated HeLa cells grown in the presence of [3H]thymidine was subjected to enzymatic hydrolysis and HPLC analysis. In control DNA, 0.3% of the thymines were modified. Thirty-six kilorads of gamma radiation caused a 30% increase in thymine damage. Thus, most of the base damage was due to internal beta radiation from incorporated [3H]thymidine. The chromatographic patterns of irradiated and nonirradiated samples were qualitatively the same, but the yields of some products increased 2-fold, while others remained unchanged. A comparison of the HPLC profiles of hydrolysates of in vitro oxidized and irradiated DNA with those of the cellular DNA revealed one fast eluting peak to be absent in cellular DNA, suggesting that it was formed only in single-stranded DNA. In cellular DNA, the major modified thymine was a more hydrophobic derivative not formed by in vitro radiation nor chemical oxidation. As in in vitro irradiated DNA, thymidine glycol constituted 5% of the modified thymines. The presence of cis-thymidine glycol in hydrolysates was confirmed by chromatography on Sephadex LH-20 using water and borate as eluants

5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) is formed in DNA by reaction with oxidizing agents and as a result of ionizing and near-ultraviolet radiation. We describe a rapid purification of cis-5,6-dihydroxy-5,6-dihydrothymine and cis-5,6-dihydroxy-5,6-dihydrothymidine (cis-thymidine glycol) and their use as markers in identifying the thymine glycol moiety in oxidized DNA. Both glycols were prepared by oxidation of [14C]thymine and -thymidine with KMnO4 followed by purification on Sephadex LH-20 (LH-20). [3H]DNA was oxidized with KMnO4 and the thymidine glycol in DNA identified by enzymatic digestion of the DNA followed by cochromatography of the digest with marker [14C]thymidine glycol on LH-20. The cis conformation of the glycol was confirmed by the change in the elution pattern when borate rather than water was used as eluent. Alkaline hydrolysis of a mixture of [14C]thymine glycol and oxidized [3H]DNA followed by trichloroacetic acid precipitation and LH-20 chromatographic analysis of the neutralized supernatant yielded a complex pattern of radioactive degradation products with coincidence of one 14C marker- and one [3H]-DNA-derived peak. All applied radioactivity was recovered. This methodology should be useful in determining thymine glycol content of irradiated DNA and in elucidating the mechanism by which these altered residues are removed from cellular DNA by repair enzymes

Two endonuclease activities in rat liver for damaged DNA were assayed. Double-stranded, covalently closed DNA from phage PM2 was damaged by either ultraviolet irradiation or by heating at acid pH, and used as substrate for endonucleases specific for ultraviolet DNA damage and for DNA apurinic sites, respectively. The levels of both enzyme activities in livers of normal rats were compared to levels in livers of rats fed N-2-acetylaminofluorene. At critical stages of the carcinogenic regimen levels of both endonuclease activities were normal. This, together with other data, suggests that depression of excision-repair of DNA damage does not take place during experimental carcinogenesis

Corrective endonuclease (correndunclease) activity of HeLa cells was assayed with alkylated DNA. Double-stranded, covalently closed DNA from phage PM II was treated with methyl methanesulfonate, N-methyl-N-nitrosourea, beta-propiolactone, or diepoxybutane to introduce alkylated bases and alkali-labile sites into the DNA. The damaged DNA was incubated with an extract of HeLa cells that catalyzes the formation of breaks at apurinic sites in double-stranded DNA. Methylated DNA was broken at every alkali-labile site by the HeLa correndonuclease, which indicated that these sites are similar to the apurinic sites produced by heating at acid pH. DNA alkylated with beta-propiolactone or diepoxybutane containing the same number of alkali-labile sites was broken to a far lesser extent. This indicates the presence of a second type of alkali-labile damage that is correndonuclease-insensitive