Base Excision Repair (BER) Glycosylase Activity Assay Service

Q: Q1: What substrates do you recommend for assaying TDG activity in the context of active demethylation?

A: For demethylation studies, we recommend duplex oligonucleotides containing a 5-carboxylcytosine (5caC) or 5-formylcytosine (5fC) paired with guanine within a CpG site context. We provide standard substrates and can synthesize custom sequences per your request.

Q: Q2: Can you analyze BER glycosylase activity from crude cell lysates or tissue samples?

A: Yes. We have optimized protocols for preparing nuclear or whole-cell extracts that preserve glycosylase activity. We include necessary controls to account for non-specific nucleases and background fluorescence in the sample.

Q: Q3: What is the minimum amount of purified enzyme required for a kinetic study?

A: For a full kinetic analysis with a purified recombinant enzyme, we typically recommend providing at least 10-20 µg of protein at a known concentration. We can work with less for initial activity confirmation; please consult with us.

Q: Q4: How do you distinguish between monofunctional (only glycosylase) and bifunctional (glycosylase/AP lyase) glycosylase activity?

A: Our standard fluorogenic assay uses a coupled enzyme (e.g., Endonuclease VIII) to cleave the AP site. To specifically detect bifunctional activity, we can omit the coupled enzyme and rely on the intrinsic β-elimination activity of the glycosylase, or use gel-based assays that differentiate the cleavage products.

Q: Q5: What deliverables do you provide for an inhibitor screening project?

A: You will receive raw fluorescence data, normalized dose-response curves, calculated IC50 values for all hits, and a ranked list of candidate inhibitors. Follow-up Ki determination and mechanism of inhibition studies are available as add-on services.

Inquiry

Base Excision Repair (BER) glycosylases are specialized enzymes that serve as the essential initiators and editors of genome integrity. CD BioSciences provides comprehensive, quantitative BER Glycosylase Activity Assay services. Utilizing state-of-the-art biochemical platforms, we deliver precise kinetic and functional profiling of key enzymes like TDG, SMUG1, and MBD4, empowering your research into genomic stability, epigenetic reprogramming, and their roles in disease pathogenesis and aging.

Introduction to BER Glycosylases

BER glycosylases are the initial and crucial enzymes in the Base Excision Repair pathway. They recognize chemically altered DNA bases, cleave the N-glycosidic bond to release the damaged base, and create an abasic site. Their primary role is to correct common DNA damage like oxidative lesions, deaminated bases, and alkylation damage. Beyond repair, a subset, notably TDG and SMUG1, participates in active DNA demethylation. TDG excises oxidized 5-methylcytosine derivatives (5fC/5caC) generated by TET enzymes, while SMUG1 removes 5hmU. This excision initiates BER-mediated restoration of unmethylated cytosine, linking genome maintenance to epigenetic regulation. Dysregulation of these enzymes is associated with cancer, neurological disorders, and aging, highlighting their biological significance.

Base Excision Repair (BER) Overview

Fig.1 Schematic Diagram of the Base Excision Repair (BER) Process. (Hindi N N, et al., 2021)

Quantifying BER glycosylase activity is vital for studying cellular responses to genotoxic stress and epigenetic dynamics. In DNA repair research, measuring enzymes like OGG1 or UNG provides a direct functional readout of a cell's ability to handle endogenous damage, serving as a biomarker for aging, neurodegeneration, and cancer risk. In epigenetics, assaying TDG activity is essential for investigating the final step of TET-driven active demethylation, as its efficiency directly impacts epigenetic reprogramming in development and differentiation. Moreover, since glycosylase activity often alters in tumors, functional assessment can reveal novel disease mechanisms.

Our Services

CD BioSciences's provides quantitative assay services to precisely characterize the function of key enzymes like TDG and SMUG1 in DNA damage repair and active demethylation. Utilizing a multi-platform approach including fluorescence, gel electrophoresis, and mass spectrometry, we perform kinetic analysis and inhibitor screening on both purified enzymes and complex biological samples.

Assays Item for Key Enzymes

TDG (Thymine-DNA Glycosylase) Activity Assay
Measures the excision efficiency of TDG against its primary substrates, including G:T mismatches and, more importantly, G:5caC or G:5fC pairs within CpG contexts, directly quantifying its role in active demethylation.
SMUG1 Activity Assay
Quantifies the glycosylase activity of SMUG1 towards substrates like 5hmU:G or U:G, assessing its function in both repair and potential demethylation pathways.
UNG, OGG1, MUTYH & Other Glycosylase Assays
We provide activity profiling for a broad range of BER glycosylases, including Uracil-DNA Glycosylase (UNG, targeting uracil), 8-Oxoguanine Glycosylase 1 (OGG1, targeting 8-oxoG), and MutY DNA Glycosylase (MUTYH, targeting A:8-oxoG mismatches).
Substrate Specificity & Kinetic Profiling
Determines enzyme preference and catalytic efficiency (Km, Vmax, kcat) across a panel of oligonucleotides containing different base lesions or sequence contexts.
Inhibitor Screening & Characterization
High-throughput or low-throughput screening services to identify and characterize small-molecule inhibitors or activators of specific BER glycosylases, complete with IC50 and Ki determination.

Service Workflow

Our project execution follows a streamlined, collaborative five-phase workflow designed for transparency, efficiency, and scientific rigor. This process ensures that your project is meticulously planned, expertly executed, and delivers actionable data on time.

Project Consultation

We discuss your specific glycosylase target, substrate of interest, and experimental goals (e.g., screening, kinetics, variant analysis).

Assay Design & Substrate Preparation

We design the optimal assay format and prepare the required lesion-containing oligonucleotide substrates, either from our stock or through custom synthesis.

Assay Optimization & Validation

The reaction conditions (buffer, pH, salt, time, enzyme concentration) are optimized for your specific enzyme-substrate pair to ensure linearity and robustness.

Sample Testing & Data Acquisition

Your provided samples (purified enzyme or cell extracts) are tested in replicate, alongside positive and negative controls, on the selected platform (fluorescent plate reader, gel scanner, or LC-MS/MS).

Data Analysis & Reporting

Data is analyzed to calculate activity rates, kinetic parameters, or inhibition values. You receive a detailed report with all experimental procedures, raw and analyzed data, and a clear summary of conclusions.

Service Applications

Mechanistic Studies of Active Demethylation

Precisely measure TDG excision kinetics on 5caC/5fC substrates to define the efficiency and sequence-context dependence of the final step in TET-mediated demethylation.

DNA Repair Capacity Biomarker Analysis

Profile the activity of OGG1, UNG, or other glycosylases in cell or tissue lysates to assess an individual's or a disease model's base excision repair capacity, relevant for aging, neurodegeneration, and cancer susceptibility studies.

Inhibitor Discovery for Novel Therapeutics

Conduct high-throughput screening campaigns to identify small molecules that selectively inhibit TDG (for potential use in oncology) or activate OGG1 (for chemoprevention), followed by detailed mechanistic characterization.

Characterization of Enzyme Variants

Characterize the activity of wild-type versus mutant or polymorphic variants of BER glycosylases (e.g., cancer-associated MUTYH variants) to determine the functional impact of genetic changes.

Deliverables

For each project, you will receive a complete data package designed for immediate analysis and reporting, including:

A comprehensive project report detailing the experimental protocol, results, and data interpretation.
All raw datasets and processed analysis files (e.g., kinetic plots, dose-response curves).
High-resolution, publication-ready figures and graphs.
Detailed kinetic parameters (Km, Vmax, kcat, IC50, Ki) where applicable.
A summary memo with expert insights and recommendations for next steps.
Ongoing technical support and consultation to discuss the findings.

Why Choose Us?

Comprehensive Glycosylase Panel. We offer activity assays for a wide spectrum of BER glycosylases (TDG, SMUG1, UNG, OGG1, MUTYH, MBD4, etc.), providing a one-stop shop for DNA repair and demethylation research.
Custom Substrate Design. We excel at synthesizing and purifying custom oligonucleotide substrates containing specific, site-specific lesions (5caC, 5fC, 5hmU, 8-oxoG, T-G mismatch, etc.), allowing you to probe enzyme specificity under biologically relevant sequence contexts.
Kinetic & Mechanistic Depth. Beyond simple endpoint activity, we provide full Michaelis-Menten kinetic analysis (Km, Vmax, kcat) and detailed mechanistic studies of inhibitors, offering deep biochemical insights.
Multi-Platform Validation. We can cross-validate key findings using orthogonal methods (e.g., fluorescence assay followed by MS analysis), ensuring the highest level of data reliability and publication readiness.
Expert Consultation on Pathway Context. Our scientists provide insights not just on the glycosylase data, but on how it integrates with upstream (TET oxidation) and downstream (AP endonuclease, polymerase, ligase) steps in the BER/demethylation pathway.

Frequently Asked Questions (FAQs)

Q1: What substrates do you recommend for assaying TDG activity in the context of active demethylation?

Q2: Can you analyze BER glycosylase activity from crude cell lysates or tissue samples?

Q3: What is the minimum amount of purified enzyme required for a kinetic study?

Q4: How do you distinguish between monofunctional (only glycosylase) and bifunctional (glycosylase/AP lyase) glycosylase activity?

Q5: What deliverables do you provide for an inhibitor screening project?

BER glycosylases are critical sentinels of genetic and epigenetic integrity. CD BioSciences provides the expert services and sophisticated platforms needed to precisely dissect their activity. Our customized, quantitative assays deliver the reliable data required to advance your research in DNA repair, active DNA demethylation, and their profound implications in health and disease. Contact us to discuss how we can tailor a BER glycosylase activity study to your specific research needs.

Reference

1. Hindi N N, Elsakrmy N, Ramotar D. The base excision repair process: comparison between higher and lower eukaryotes[J]. Cellular and Molecular Life Sciences, 2021, 78(24): 7943-7965.