https://scholars.lib.ntu.edu.tw/handle/123456789/618543
Title: | Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis | Authors: | Chang, Hui-Lan KANG-YI SU Goodman, Steven D WERN-CHERNG CHENG LIANG-IN LIN YA-CHIEN YANG SUI-YUAN CHANG WOEI-HORNG FANG |
Keywords: | ESCHERICHIA-COLI; AMPLIFICATION STRATEGY; SINGLE-NUCLEOTIDE; EXCISION; REPAIR; BASE; SUBSTRATE; CELLS; DEAMINATION; GLYCOSIDASE | Issue Date: | 2022 | Publisher: | JOURNAL OF VISUALIZED EXPERIMENTS | Journal Volume: | 2022 | Journal Issue: | 182 | Source: | Journal of visualized experiments : JoVE | Abstract: | Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening. |
URI: | https://scholars.lib.ntu.edu.tw/handle/123456789/618543 | ISSN: | 1940-087X | DOI: | 10.3791/63089 |
Appears in Collections: | 醫學檢驗暨生物技術學系 |
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