Ganguly T, Duker N J
Department of Pathology, Temple University School of Medicine, Philadelphia, PA 19140.
Nucleic Acids Res. 1991 Jun 25;19(12):3319-23. doi: 10.1093/nar/19.12.3319.
Pyrimidine hydrates are products of ultraviolet irradiation of DNA. We have already demonstrated the formation of both cis-thymine hydrate and trans-thymine hydrate (6-hydroxy-5,6-dihydrothymine) in irradiated poly(dA-dT):poly(dA-dT). These are released from DNA as free bases by bacterial or human glycosylases. Thymine hydrate stabilities were studied in irradiated DNA substrates using purified E. coli endonuclease III as a reagent for their removal. After irradiation, substrate poly(dA-dT):poly(dA-dT), radiolabeled in thymine, was incubated at 50, 60, 70 or 80 degrees C, cooled, and then reacted with the enzyme under standard conditions. Thymine hydrates were assayed by enzymic release of labeled material into the ethanol-soluble fraction. Their identities were confirmed by high performance liquid chromatography. The decay of thymine hydrates in heated DNA followed first-order kinetics with a k = 2.8 x 10(-5)/sec at 80 degrees C. These hydrates were also detected in lesser quantities in the unirradiated, control substrate. Extrapolation from an Arrhenius plot yields an estimated half-life of 33.3 hours at 37 degrees C for DNA thymine hydrates. Such stability, together with their formation in unirradiated DNA, suggest thymine hydrates to be formed under physiological conditions and to be sufficiently stable in DNA to be potentially genotoxic. This necessitates their constant removal from DNA by the excision-repair system.
嘧啶水合物是DNA紫外线照射的产物。我们已经证实在经照射的聚（dA-dT）：聚（dA-dT）中会形成顺式胸腺嘧啶水合物和反式胸腺嘧啶水合物（6-羟基-5,6-二氢胸腺嘧啶）。这些水合物通过细菌或人类糖苷酶从DNA中以游离碱基的形式释放出来。使用纯化的大肠杆菌内切核酸酶III作为去除胸腺嘧啶水合物的试剂，研究了经照射的DNA底物中胸腺嘧啶水合物的稳定性。照射后，在胸腺嘧啶中进行放射性标记的底物聚（dA-dT）：聚（dA-dT）在50、60、70或80摄氏度下孵育，冷却，然后在标准条件下与酶反应。通过将标记物质酶促释放到乙醇可溶级分中来测定胸腺嘧啶水合物。通过高效液相色谱法确认它们的身份。加热的DNA中胸腺嘧啶水合物的衰变遵循一级动力学，在80摄氏度下k = 2.8×10⁻⁵/秒。在未照射的对照底物中也检测到了少量的这些水合物。从阿累尼乌斯图外推得出，DNA胸腺嘧啶水合物在37摄氏度下的估计半衰期为33.3小时。这种稳定性以及它们在未照射的DNA中的形成表明，胸腺嘧啶水合物是在生理条件下形成的，并且在DNA中足够稳定，可能具有基因毒性。这就需要切除修复系统不断地将它们从DNA中去除。
