Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

Summary

A non-labeled, non-radio-isotopic method to assay uracil-DNA glycosylase activity was developed using MALDI-TOF mass spectrometry for direct apurinic/apyrimidinic site-containing product analysis. The assay proved to be quite simple, specific, rapid, and easy to use for DNA glycosylase measurement.

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 K_m = 50 nM, V_max = 0.98 nM/s, and K_cat = 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.

Introduction

Although uracil is a normal base in RNA, it is a common and highly mutagenic lesion in genomic DNA. Uracil can arise from spontaneous/enzymatic hydrolytic deamination of a deoxycytidine. In each living cell, this deamination occurs 100-500 times per day under physiological conditions^1,2. If these alterations are not repaired, there can be a change in the DNA sequence composition, causing mutation. As uracil in DNA prefers to pair with dATP during replication, if cytosine deaminates to uracil, in two replication events, there will be a new G:C to A:T transition mutation in half of the progeny DNA

Protocol

1. Substrate/template preparation

1. Design uracil substrate/template duplex with a balanced G+C content of ~50 ± 10% and minimum melting temperature of 50 °C for the duplex region.
   NOTE: One nucleotide difference between 18 nt substrates and 19 nt templates (Table 1 and Figure 1) helps better MS signal interpretation and appropriate annealing. The template strand serves as complementary DNA to generate A-U or G-U mismatches (Table 1) but can also be used as a reference signal in MS measurements. The use of HPLC-purified synthetic oligonucleotides is satisfactory....

Results

Templates and substrates
Taking synthetic oligonucleotides with U in the center (U+9) paired with a G template as an example (Figure 1A), a blank control of equimolar amounts of template and uracil-containing substrate can be used for quality control of synthetic oligonucleotide purity (Figure 1B; the signals match the designated m/z and the low background noise). For the MS data analysis, the peak heights were measured (

Discussion

This paper provides a detailed procedure for using a UDG MALDI-TOF MS assay method to directly detect AP-containing DNA products. The main advantages of this method are that uracil-containing substrates are label-free, scalable, easy to work with, and afford greater flexibility in substrate design.

The UDG supplier-recommended phenol/chloroform extraction enables inactivation of the enzyme to prevent degradation of product DNA. However, the phenol extraction protocol involves tedious phase-sep.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris base)	J.T baker		Protocol 1,2
Autoclaved deionized water	MILLIPORE		Protocol 1,2
EDTA	J.T Baker		Protocol 1,2
Gloves	AQUAGLOVE		Protocol 1,2,3
Hydrochloric acid (HCl)	SIGMA		Protocol 1,2
Ice bucket	Taiwan.Inc		Protocol 2
Low retention pipette tips(0.5-10 µL)	extra gene		Protocol 1,2
Low retention pipette tips(1,250 µL)	national scientific supply co, Inc.		Protocol 1,2
Low retention pipette tips(200 µL)	national scientific supply co, Inc.		Protocol 1,2
MassARRAY	Agena Bioscience, CA		Protocol 4, 5 Mass spectrometry control programs include Typer Chip Linker, SpectroACQUIRE, and Start RT Process.
MassARRAY Nanodispenser	AAT Bioquest, Inc.	RS1000	Protocol 3
Microcentrifuge	Kubota		Protocol 2
Microcentrifuge	Clubio		Protocol 2
Microcentrifuge tube (1.5 mL)	National scientific supply co, Inc.		Protocol 2
Microcentrifuge tube rack	Taiwan.Inc		Protocol 1,2
Micropipette  (P1000)	Gilson		Protocol 1,2
Micropipette  (P2)	Gilson		Protocol 1,2
Micropipette (P10)	Gilson		Protocol 1,2
Micropipette (P100)	Gilson		Protocol 1,2
Micropipette (P200)	Gilson		Protocol 1,2
Micropipette (SL2)	Rainin		Protocol 1,2
Oligonucleotides	Integrated DNA Technologies (Singapore)		Protocol 1,2
Quest Graph IC50 Calculator (v.1)	AAT Bioquest, Inc.		Fig. 4 https://www.aatbio.com/tools/ic50-calculator-v1
Sodium hydroxide (NaOH)	WAKO		Protocol 2
SpectroCHIP array	Agena Bioscience, CA	#01509	Protocol 3, 5
Timer	Taiwan.Inc		Protocol 2
Typer 4.0 software	Agena Bioscience, CA	#10145	Protocol 6 Typer 4.0 consists four programs including Assay Designer, Assay Editor, Plate Editor, and Typer Analyzer.
UDG Reaction Buffer (10x)	New England Biolabs, MA	B0280S	Protocol 2
Uracil Glycosylase Inhibitor	New England Biolabs, MA	M0281S	Protocol 2
Uracil-DNA Glycosylase	New England Biolabs, MA	M0280L	Protocol 2
UV-VISBLE spectrophotometer UV-1601	SHIMADZU		Protocol 1
Water bath	ZETA ZC-4000 (Taiwan.Inc)		Protocol 2