Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, USA; MD-PhD Combined Degree Program, University of Texas Medical Branch, Galveston, Texas, USA.
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, USA.
J Biol Chem. 2022 Mar;298(3):101638. doi: 10.1016/j.jbc.2022.101638. Epub 2022 Jan 25.
The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems, and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase (TDG) that is comprised of a 29-amino acid sequence from human TDG linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hybrid TDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC-MS/MS quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts.
胞嘧啶和 5-甲基胞嘧啶的水解脱氨作用驱动了许多在人类癌症中观察到的转换突变。脱氨诱导的诱变中间体包括与鸟嘌呤错配的尿嘧啶或胸腺嘧啶加合物。虽然存在大量的方法可以测量其他类型的 DNA 加合物，但胞嘧啶脱氨加合物带来了不寻常的分析问题，并且尚未开发出充分的方法来测量它们。我们在这里描述了一种新型的混合胸腺嘧啶 DNA 糖基化酶（TDG），它由人类 TDG 的 29 个氨基酸序列与在一种古细菌嗜热菌中发现的胸腺嘧啶糖苷酶的催化结构域连接而成。使用确定序列的寡核苷酸，我们表明杂交 TDG 对脱氨的 U:G 和 T:G 错配具有强大的错配选择性活性。我们进一步开发了一种从寡核苷酸和 DNA 中分离糖苷酶释放的游离碱基的方法，然后进行 GC-MS/MS 定量。使用这种方法，我们首次测量了小牛胸腺 DNA 中总尿嘧啶、U:G 和 T:G 对的水平。这里提出的方法将允许测量生物上重要的一类脱氨胞嘧啶加合物的形成、持续存在和修复。
