DNA Glycosylase Activity Assay Based on Streptavidin Paramagnetic Bead Substrate Capture

doi:10.1006/abio.2001.5400

Analytical Biochemistry

Volume 298, Issue 2, 15 November 2001, Pages 322-326

https://doi.org/10.1006/abio.2001.5400 Get rights and content

Abstract

A method to detect DNA glycosylase activity is described. The substrate used was an oligodeoxyribonucleotide with a unique hypoxanthine base, but has general application to any DNA glycosylase or endonuclease. The oligodeoxyribonucleotide was labeled at the 5′ end with ³²P and at the 3′ end with a biotin linkage and annealed to a complementary oligodeoxyribonucleotide. The hypoxanthine base was excised in solution using the MPG protein, a human DNA glycosylase. Following cleavage of the phosphodiester linkage by NaOH, the oligodeoxyribonucleotide was attached to streptavidin-coated, paramagnetic beads. Binding of the labeled oligodeoxyribonucleotide to the beads was indicative of the kinetics of the reaction. As a control, half of the reaction products were loaded on to a denaturing polyacrylamide gel. Comparable values for steady-state kinetic constants were obtained using both methods. This nonelectrophoretic technique is a rapid assay of DNA glycosylase activity for both purified proteins and crude extracts. This method can be directly adapted for high-throughput techniques.

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Cited by (31)

Construction of a target-triggered DNAzyme motor for electrochemical detection of multiple DNA glycosylases
2022, Sensors and Actuators B: Chemical
DNA repair enzymes are responsible for the repair of DNA lesions and are important disease biomarkers. Herein, we construct a target-triggered DNAzyme motor for electrochemical detection of multiple DNA glycosylases based on the DNAzyme motor-driven encoding of different electrochemical molecules. This assay involves two hairpin probes for sensing uracil DNA glycosylase (UDG) and human alkyladenine DNA glycosylase (hAAG), a AuNPs@locked DNAzyme motor which contains the locking strands and the Mg²⁺/Pb²⁺ DNAzyme strands, and a magnetic bead track which contains MB/Fc-labelled Mg²⁺/Pb²⁺ substrate strands. When UDG and hAAG are present, they can cleave the corresponding hairpin DNA probes to release two single-stranded DNAs (ssDNAs) which can hybridize with the locking strands to induce the liberation of Mg²⁺/Pb²⁺ DNAzyme strands from the AuNPs@DNAzyme motor. The subsequent hybridization of Mg²⁺/Pb²⁺ DNAzyme strands with their corresponding substrate strands results in the cleavage of Mg²⁺/Pb²⁺ substrate strands and the simultaneous release of MB/Fc-labelled ssDNAs from magnetic bead. The released MB/Fc labels can be electrochemically detected for accurate quantification of UDG/hAAG. This target-triggered DNAzyme motor exhibits high sensitivity and good specificity with the capability of simultaneously measuring multiple DNA glycosylases in HeLa cells and screening the potential inhibitors.
Recent advances in biosensor for DNA glycosylase activity detection
2022, Talanta
Citation Excerpt :
Up to the present, lots of strategies have been presented to monitor the activity of DNA glycosylase, especially UDG. Several traditional methodologies, among which are mass spectrometry [12], streptavidin paramagnetic bead capture method [13], radio-isotopic labeling [14], and gel electrophoresis [15], have been utilized to detect DNA glycosylase activity. However, because of disadvantages like indirectness, insensitivity, and complicated operation, conventional methods of DNA glycosylase activity measurements seem far from sufficiency.
Base excision repair (BER) is vital for maintaining the integrity of the genome under oxidative damage. DNA glycosylase initiates the BER pathway recognizes and excises the mismatched substrate base leading to the apurinic/apyrimidinic site generation, and simultaneously breaks the single-strand DNA. As the aberrant activity of DNA glycosylase is associated with numerous diseases, including cancer, immunodeficiency, and atherosclerosis, the detection of DNA glycosylase is significant from bench to bedside. In this review, we summarized novel DNA strategies in the past five years for DNA glycosylase activity detection, which are classified into fluorescence, colorimetric, electrochemical strategies, etc. We also highlight the current limitations and look into the future of DNA glycosylase activity monitoring.
Combination of bidirectional strand displacement amplification with single-molecule detection for multiplexed DNA glycosylases assay
2021, Talanta
Citation Excerpt :
Notably, multiple DNA glycosylases exhibit abnormal in some diseases [8], and thus the simultaneous measurement of different DNA glycosylases is essential to clinical diagnosis and biochemical research. Traditional techniques used for DNA glycosylase assay include liquid chromatography/isotope-dilution tandem mass spectrometry [14], high-performance liquid chromatography [15], gel electrophoresis [16], and paramagnetic bead capturing technique [17]. However, these assays are restrained by the involvement of hazardous radioactive labelling, sophisticated instrumentation, complicated manipulation procedures, time-consuming preparation, and insufficient sensitivity [14–18].
DNA glycosylases can initiate base excision repair pathway to repair endogenous DNA base damages for the maintenance of genome stability. Multiple DNA glycosylases exhibit abnormal in various diseases, and the simultaneous measurement of different DNA glycosylases is critical to clinical diagnosis and drug discovery. Herein, we take advantage of single-molecule detection and bidirectional strand displacement amplification (SDA) to simultaneously detect uracil DNA glycolase (UDG) and human alkyladenine DNA glycosylase (hAAG). We design a partial double-stranded DNA (dsDNA) substrate modified with specific recognition sites of UDG and hAAG. The dsDNA substrate is labeled with BHQ1 and BHQ2 at the 5′-ends and then hybridizes with the Cy3/Cy5-labeled reporter probes to obtain the BHQ1/Cy3 and BHQ2/Cy5 base pairs, resulting in the quenching of Cy3/Cy5 fluorescence by BHQ1/BHQ2 via ﬂuorescence resonance energy transfer (FRET). When UDG and hAAG are present, they can induce the base excision repair reaction and subsequently initiate the bidirectional SDA amplification process, releasing the Cy5/Cy3-labeled reporter probes from the dsDNA substrate and consequently the recovery of Cy5 and Cy3 fluorescence, which can be measured by single-molecule detection, with Cy5 indicating UDG and Cy3 indicating hAAG. This method possesses high sensitivity and good selectivity with the capability of quantifying multiple DNA glycosylases at the single-cell level. Furthermore, it can be used to simultaneously screen DNA glycosylase inhibitors and determine enzyme kinetic parameters, with the potential of sensing various DNA/RNA enzymes by simple changing the recognition sites of DNA substrates.
Measurement of uracil-DNA glycosylase activity by matrix assisted laser desorption/ionization time-of-flight mass spectrometry technique
2021, DNA Repair
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.
Integrating DNA structure switch with branched hairpins for the detection of uracil-DNA glycosylase activity and inhibitor screening
2018, Talanta
The detection of uracil-DNA glycosylase (UDG) activity is pivotal for its biochemical studies and the development of drugs for UDG-related diseases. Here, we explored an integrated DNA structure switch for high sensitive detection of UDG activity. The DNA structure switch containing two branched hairpins was employed to recognize UDG enzyme and generate fluorescent signal. Under the action of UDG, one branched hairpin was impelled folding into a close conformation after the excision of the single uracil. This reconfigured hairpin could immediately initiate the polymerization/nicking amplification reaction of another branched hairpin accompanying with the release of numerous G-quadruplexes (G4s). In the absence of UDG, the DNA structure switch kept its original configuration, and thus the subsequent polymerization/nicking reaction was inhibited, resulting in the release of few G4 strands. In this work, Thioflavin T was used as signal reporter to target G4s. By integrating the DNA structure switch, the quick response and high sensitivity for UDG determination was achieved and a low detection limit of 0.0001 U/mL was obtained, which was superior to the most fluorescent methods for UDG assay. The repeatability of the as-proposed strategy was demonstrated under the concentration of 0.02 U/mL and 0.002 U/mL, the relative standard deviation obtained from 5 successive samples were 1.7% and 2.8%, respectively. The integrated DNA structure switch strategy proposed here has the potential application for the study of mechanism and function of UDG enzyme and the screening the inhibitors as potential drugs and biochemical tools.
Tungsten disulfide nanosheet and exonuclease III co-assisted amplification strategy for highly sensitive fluorescence polarization detection of DNA glycosylase activity
2015, Analytica Chimica Acta
Citation Excerpt :
Accordingly, the monitoring DNA glycosylase activity plays an important role in the study of repair mechanism and development of tools for molecular diagnostics. Gel electrophoresis is the traditional detection methods for DNA glycosylase activity [26,27], which is time-consuming, complicated and of insufficient sensitivity. Consequently, several novel detection methods have been developed to overcome these limits, such as fluorescence resonant energy transfer based detection methods [28–31], G-quadruplex based fluorescent approaches [32] and gold nanoparticle based colorimetric assay [33,34].
Herein, we introduced a tungsten disulfide (WS₂) nanosheet and exonuclease III (Exo III) co-assisted signal amplification strategy for highly sensitive fluorescent polarization (FP) assay of DNA glycosylase activity. Two DNA glycosylases, uracil-DNA glycosylase (UDG) and human 8-oxoG DNA glycosylase 1 (hOGG1), were tested. A hairpin-structured probe (HP) which contained damaged bases in the stem was used as the substrate. The removal of damaged bases from substrate by DNA glycosylase would lower the melting temperature of HP. The HP was then opened and hybridized with a FAM dye-labeled single strand DNA (DP), generating a duplex with a recessed 3′-terminal of DP. This design facilitated the Exo III-assisted amplification by repeating the hybridization and digestion of DP, liberating numerous FAM fluorophores which could not be adsorbed on WS₂ nanosheet. Thus, the final system exhibited a small FP signal. However, in the absence of DNA glycosylases, no hybridization between DP and HP was occurred, hampering the hydrolysis of DP by Exo III. The intact DP was then adsorbed on the surface of WS₂ nanosheet that greatly amplified the mass of the labeled-FAM fluorophore, resulting in a large FP value. With the co-assisted amplification strategy, the sensitivity was substantially improved. In addition, this method was applied to detect UDG activity in cell extracts. The study of the inhibition of UDG was also performed. Furthermore, this method is simple in design, easy in implementation, and selective, which holds potential applications in the DNA glycosylase related mechanism research and molecular diagnostics.

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¹: To whom correspondence and reprint requests should be addressed. Fax: (626) 930-5366. E-mail: [email protected].

²: On leave from CNRS UMR1772 Physicochimie et Pharmacologie des Macromolecules Biologiques.

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Regular ArticleDNA Glycosylase Activity Assay Based on Streptavidin Paramagnetic Bead Substrate Capture

Abstract

Mutat. Res.

FEBS Lett.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Cell

Quality control by DNA repair

Science

Initiation of base excision repair: Glycosylase mechanisms and structures

Annu. Rev. Biochem.

Release of 7-methylguanine residues whose imidazole rings have been opened from damaged DNA by a DNA glycosylase from Escherichia coli

Nucleic Acids Res.

Two enzymes are required from strand incision in repair of alkylated DNA

Nature

An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues

Proc. Natl. Acad. Sci. USA

New class of enzymes acting on damaged DNA

Nature

Properties of 3-methyladenine–DNA glycosylase from Escherichia coli

Biochemistry

Assays for the repair of oxidative damage by formamidopyrimidine glycosylase (Fpg) and 8-oxoguanine DNA glycosylase (OGG-1)

Methods Mol. Biol.

Purification and characterization of human 3-methyladenine–DNA glycosylase

Nucleic Acids Res.

Regular Article
DNA Glycosylase Activity Assay Based on Streptavidin Paramagnetic Bead Substrate Capture