D'Ham C, Ravanat J L, Cadet J
Département de Recherche Fondamentale sur la Matière Condensée/SCIB, Laboratoire Lésions des Acides Nucléiques, CEA/Grenoble, France.
J Chromatogr B Biomed Sci Appl. 1998 Jun 12;710(1-2):67-74. doi: 10.1016/s0378-4347(98)00133-9.
The endonuclease III from Escherichia coli is a repair enzyme which exhibits both a glycosylase and an endonuclease function. The activity of the enzyme can be assayed by measuring the released targeted bases in solution from a sample of modified DNA. In the present study, gas chromatography-mass spectrometry was used together with an HPLC prepurification step in order to single out the released bases. The prepurification was found to enhance the specificity and the sensitivity of the assay. Thus, the overall method allowed us to analyze separately 5-hydroxy-5,6-dihydrothymine from the cis and trans isomers of 6-hydroxy-5,6-dihydrothymine. Examples of application of the assay are provided with the measurement of the E. coli endonuclease III-mediated excision of 5-hydroxy-5,6-dihydrothymine and 5,6-dihydrothymine from samples of gamma-irradiated DNA in the presence of cysteine.
来自大肠杆菌的核酸内切酶III是一种修复酶，它兼具糖基化酶和核酸内切酶的功能。该酶的活性可以通过测量从修饰DNA样品中释放到溶液中的目标碱基来测定。在本研究中，气相色谱-质谱联用高效液相色谱预纯化步骤，以分离出释放的碱基。发现预纯化可提高测定的特异性和灵敏度。因此，该整体方法使我们能够分别分析5-羟基-5,6-二氢胸腺嘧啶与6-羟基-5,6-二氢胸腺嘧啶的顺式和反式异构体。文中给出了该测定方法的应用实例，即测量在半胱氨酸存在下，大肠杆菌核酸内切酶III介导的从γ射线辐照DNA样品中切除5-羟基-5,6-二氢胸腺嘧啶和5,6-二氢胸腺嘧啶的情况。
