DNA Repair Molecular Beacon assay: a platform for real-time functional analysis of cellular DNA repair capacity
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Jianfeng Li1, David Svilar2,3, Steven McClellan1, Jung-Hyun Kim1, Eun-Young Erin Ahn1, Conchita Vens4, David M. Wilson III5 and Robert W. Sobol1,2,3
1University of South Alabama Mitchell Cancer Institute, Mobile, AL, USA
2Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
3University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, PA, USA
4The Netherlands Cancer Institute, Division of Cell Biology, Amsterdam, The Netherlands
5Laboratory of Molecular Gerontology, National Institute on Aging, IRP, NIH Baltimore, MD, USA
Robert W. Sobol, email: [email protected]
Keywords: base excision repair; molecular beacon assay; microbead-conjugated molecular beacon assay; DNA glycosylase; APE1
Received: February 14, 2018 Accepted: July 12, 2018 Published: August 03, 2018
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 application. Our studies establish an analytical platform capable of evaluating the enzyme activity of select DNA repair proteins in an effort to design and guide inhibitor development and precision cancer therapy options.
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