通过高压液相色谱分析鉴定化学氧化和γ射线辐照的脱氧核糖核酸中的顺式胸腺嘧啶乙二醇部分。

Identification of the cis-thymine glycol moiety in chemically oxidized and gamma-irradiated deoxyribonucleic acid by high-pressure liquid chromatography analysis.

作者信息

Frenkel K, Goldstein M S, Teebor G W

出版信息

Biochemistry. 1981 Dec 22;20(26):7566-71. doi: 10.1021/bi00529a035.

Abstract

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.

摘要

5,6 - 二羟基 - 5,6 - 二氢胸腺嘧啶（胸腺嘧啶二醇）由化学氧化剂和电离辐射在DNA中形成。我们描述了通过高压液相色谱法（HPLC）分离胸腺嘧啶二醇、5,6 - 二羟基 - 5,6 - 二氢胸苷（胸苷二醇）、胸腺嘧啶和胸苷。将化学氧化或γ射线辐照的单链DNA的酶促水解产物与含14C的标记化合物进行共色谱分析。在化学氧化的DNA中，胸苷二醇是形成的主要衍生物。此外，还有四种快速洗脱的胸腺嘧啶衍生成分。在辐照的DNA中，胸苷二醇占修饰胸腺嘧啶的约5%，并且快速洗脱部分相应增加。从在[3H]胸腺嘧啶存在下生长的γ射线辐照和未辐照的HeLa细胞中分离的DNA进行酶促水解和HPLC分析。在对照DNA中，0.3%的胸腺嘧啶被修饰。36千拉德的γ射线辐射导致胸腺嘧啶损伤增加30%。因此，大部分碱基损伤是由于掺入的[3H]胸腺嘧啶的内源性β辐射。辐照和未辐照样品的色谱图在定性上相同，但一些产物的产量增加了2倍，而其他产物保持不变。将体外氧化和辐照的DNA水解产物的HPLC图谱与细胞DNA的图谱进行比较，发现细胞DNA中不存在一个快速洗脱峰，这表明它仅在单链DNA中形成。在细胞DNA中，主要的修饰胸腺嘧啶是一种疏水性更强的衍生物，体外辐射和化学氧化均未形成这种衍生物。与体外辐照的DNA一样，胸苷二醇占修饰胸腺嘧啶的5%。通过使用水和硼酸盐作为洗脱剂在Sephadex LH - 20上进行色谱分析，证实了水解产物中顺式胸苷二醇的存在。