DNA Repair Molecular Beacon assay: a platform for real-time functional analysis of cellular DNA repair capacity

Oncotarget
Jianfeng LiRobert W Sobol

Abstract

Numerous studies have shown that select DNA repair enzyme activities impact response and/or toxicity of genotoxins, suggesting a requirement for enzyme functional analyses to bolster precision medicine or prevention. To address this need, we developed a DNA Repair Molecular Beacon (DRMB) platform that rapidly measures DNA repair enzyme activity in real-time. The DRMB assay is applicable for discovery of DNA repair enzyme inhibitors, for the quantification of enzyme rates and is sufficiently sensitive to differentiate cellular enzymatic activity that stems from variation in expression or effects of amino acid substitutions. We show activity measures of several different base excision repair (BER) enzymes, including proteins with tumor-identified point mutations, revealing lesion-, lesion-context- and cell-type-specific repair dependence; suggesting application for DNA repair capacity analysis of tumors. DRMB measurements using lysates from isogenic control and APE1-deficient human cells suggests the major mechanism of base lesion removal by most DNA glycosylases may be mono-functional base hydrolysis. In addition, development of a microbead-conjugated DRMB assay amenable to flow cytometric analysis further advances its applicati...Continue Reading

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Citations

Mar 11, 2020·Cancer Epidemiology, Biomarkers & Prevention : a Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology·Martyn T SmithMark Fielden
May 27, 2020·DNA Repair·Amy M WhitakerBret D Freudenthal
Jul 3, 2021·Biology·Marlo K ThompsonAishwarya Prakash
Aug 9, 2021·DNA Repair·Jing GeBevin P Engelward
Aug 28, 2021·International Journal of Molecular Sciences·Marina Jordano-RayaDolores Córdoba-Cañero

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Methods Mentioned

BETA
glycosylases
flow cytometry
transfection
glycosylase
Protein Extraction
protein assay
gel-filtration
Flow

Software Mentioned

GraphPad Prism
DRMB
MUTYH

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