Measurement of uracil-DNA glycosylase activity by matrix assisted laser desorption/ionization time-of-flight mass spectrometry technique

Hui-Lan Chang; Kang-Yi Su; Steven D Goodman; Rong-Syuan Yen; Wern-Cherng Cheng; Ya-Chien Yang; Liang-In Lin; Sui-Yuan Chang; Woei-Horng Fang

doi:10.1016/j.dnarep.2020.103028

Measurement of uracil-DNA glycosylase activity by matrix assisted laser desorption/ionization time-of-flight mass spectrometry technique

DNA Repair (Amst). 2021 Jan:97:103028. doi: 10.1016/j.dnarep.2020.103028. Epub 2020 Nov 24.

Authors

Hui-Lan Chang¹, Kang-Yi Su², Steven D Goodman³, Rong-Syuan Yen¹, Wern-Cherng Cheng⁴, Ya-Chien Yang², Liang-In Lin², Sui-Yuan Chang², Woei-Horng Fang⁵

Affiliations

¹ Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan, ROC.
² Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan, ROC; Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan, ROC.
³ Center for Microbial Pathogenesis, Nationwide Children's Hospital and the Department of Pediatrics, the Ohio State University, Columbus, OH, USA.
⁴ Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan, ROC.
⁵ Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan, ROC; Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan, ROC. Electronic address: whfang@ntu.edu.tw.

PMID: 33254084
DOI: 10.1016/j.dnarep.2020.103028

Abstract

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.

Keywords: Apurinic/apyrimidinic site; Base excision repair; MALDI-TOF MS; Uracil-DNA glycosylase.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA Damage
DNA Repair*
DNA, Bacterial / metabolism*
Escherichia coli / enzymology*
Escherichia coli / genetics
Kinetics
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization*
Uracil / analysis*
Uracil / metabolism
Uracil-DNA Glycosidase / metabolism*

Substances

DNA, Bacterial
Uracil
Uracil-DNA Glycosidase