Uracil-DNA glycosylase (UDG) is a highly conserved DNA repair enzyme that acts as a key component in the base excision repair pathway to correct hydrolytic deamination of cytosine making it critical to genome integrity in living organisms. We report here a non-labeled, non-radio-isotopic and very specific method to measure UDG activity. Oligodeoxyribonucleotide duplex containing a site-specific G:U mismatch that is hydrolyzed by UDG then subjected to Matrix Assisted Laser Desorption/Ionization time-of-flight mass spectrometry analysis. A protocol was developed to maintain the AP product in DNA without strand break then the cleavage of uracil was identified by the mass change from uracil substrate to AP product. From UDG kinetic analysis, for G:U substrate the K_m is 50 nM, V_max is 0.98 nM/s and K_cat = 9.31 s⁻¹. The method was applied to uracil glycosylase inhibitor measurement with an IC₅₀ value of 7.6 pM. Single-stranded and double-stranded DNAs with uracil at various positions of the substrates were also tested for UDG activity albeit with different efficiencies. The simple, rapid, quantifiable, scalable and versatile method has potential to be the reference method for monofunctional glycosylase measurement, and can also be used as a tool for glycosylase inhibitors screening.

尿嘧啶-DNA 糖基化酶 (UDG) 是一种高度保守的 DNA 修复酶，是碱基切除修复途径中的关键成分，可纠正胞嘧啶的水解脱氨，使其对生物体的基因组完整性至关重要。我们在此报告了一种非标记、非放射性同位素且非常具体的测量 UDG 活性的方法。寡聚脱氧核糖核苷酸双链体含有位点特异性 G:U 错配，由 UDG 水解，然后进行基质辅助激光解吸/电离飞行时间质谱分析。开发了一种方案以保持 DNA 中的 AP 产物没有链断裂，然后通过从尿嘧啶底物到 AP 产物的质量变化来鉴定尿嘧啶的切割。根据 UDG 动力学分析，对于 G:U 底物，K _m为 50 nM，V_最大值为 0.98 nM/s 且K _cat = 9.31 s ^-1。该方法应用于尿嘧啶糖基化酶抑制剂测量，IC ₅₀值为7.6 pM。尽管效率不同，但还测试了在底物不同位置具有尿嘧啶的单链和双链 DNA 的 UDG 活性。这种简单、快速、可量化、可扩展和通用的方法有可能成为单功能糖基化酶测量的参考方法，也可用作糖基化酶抑制剂筛选的工具。