Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis
Name
Nagel-2014-Multiplexed DNA repair.pdf
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1.38 MB
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Checksum (MD5)
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Author(s)
Nagel, Zachary D.
Margulies, Carrie Marie
Chaim, Isaac Alexander
McRee, Siobhan K.
Mazzucato, Patrizia
Ahmad, Anwaar
Abo, Ryan
Butty, Vincent
Forget, Anthony L.
Samson, Leona D.
Date Issued
April 2014
Journal
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences (U.S.)
Citation
Nagel, Z. D., C. M. Margulies, I. A. Chaim, S. K. McRee, P. Mazzucato, A. Ahmad, R. P. Abo, V. L. Butty, A. L. Forget, and L. D. Samson. “Multiplexed DNA Repair Assays for Multiple Lesions and Multiple Doses via Transcription Inhibition and Transcriptional Mutagenesis.” Proceedings of the National Academy of Sciences 111, no. 18 (April 22, 2014): E1823–E1832.
Version
Final published version
Abstract
The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for measuring DNA repair capacity (DRC) are relatively labor intensive, often indirect, and usually limited to a single repair pathway. Here, we describe a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) that measures the ability of human cells to repair plasmid reporters, each bearing a different type of DNA damage or different doses of the same type of DNA damage. FM-HCR simultaneously measures repair capacity in any four of the following pathways: nucleotide excision repair, mismatch repair, base excision repair, nonhomologous end joining, homologous recombination, and methylguanine methyltransferase. We show that FM-HCR can measure interindividual DRC differences in a panel of 24 cell lines derived from genetically diverse, apparently healthy individuals, and we show that FM-HCR may be used to identify inhibitors or enhancers of DRC. We further develop a next-generation sequencing-based HCR assay (HCR-Seq) that detects rare transcriptional mutagenesis events due to lesion bypass by RNA polymerase, providing an added dimension to DRC measurements. FM-HCR and HCR-Seq provide powerful tools for exploring relationships among global DRC, disease susceptibility, and optimal treatment.
MIT Department
Massachusetts Institute of Technology. Center for Environmental Health Sciences
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Biology
Koch Institute for Integrative Cancer Research at MIT
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DOI of Published Version
https://doi.org/10.1073/pnas.1401182111
