Abstract
DNA double-strand breaks (DSBs) are the most lethal genomic lesions that are induced endogenously during physiological reactions as well as by external stimuli and genotoxicants. DSBs are repaired in mammalian cells via one of three well-studied pathways depending on the cell cycle status and/or the nature of the break. First, the homologous recombination (HR) pathway utilizes the duplicated sister chromatid as a template in S/G2 cells. Second, the nonhomologous end-joining (NHEJ) is the predominant DSB repair pathway throughout the cell cycle. The third pathway, microhomology-mediated/alternative end-joining (MMEJ/Alt-EJ), is a specialized backup pathway that works not only in the S phase but also in G0/G1 cells that constitute the bulk of human tissues. In vitro experimental methods to recapitulate the repair of physiologically relevant DSBs pose a challenge. Commonly employed plasmid- or oligonucleotide-based substrates contain restriction enzyme-cleaved DSB mimics, which undoubtedly do not mimic DSB ends generated by ionizing radiation (IR), chemotherapeutics, and reactive oxygen species (ROS). DSBs can also be indirectly generated by reactive oxygen species (ROS). All such DSBs invariably contain blocked termini. In this methodology chapter, we describe a method to recapitulate the DSB repair mechanism using in cellulo and in vitro cell-free systems. This methodology enables researchers to assess the contribution of NHEJ vs. Alt-EJ using a reporter plasmid containing DSB lesions with non-ligatable termini. Limitations and challenges of prevailing methods are also addressed.
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Acknowledgments
Research in the authors’ laboratory is supported by grants from the National Institute of Neurological Disorders and Stroke (NINDS) and the National Institute of Aging (NIA) of the National Institutes of Health (NIH) under award numbers R01NS088645, RF1NS112719, R03AG064266, and R01NS094535 and the Houston Methodist Research Institute’s internal funds. M.L.H. thanks Everett E. and Randee K. Bernal for their support via the Centenial Endowed Directorship of DNA Repair. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies. The authors thank Dr. Gillian Hamilton at Houston Methodist Research Institute (Houston, TX) for her assistance with editing the document.
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Dutta, A., Mitra, J., Hegde, P.M., Mitra, S., Hegde, M.L. (2023). Characterizing the Repair of DNA Double-Strand Breaks: A Review of Surrogate Plasmid-Based Reporter Methods. In: Bhakat, K.K., Hazra, T.K. (eds) Base Excision Repair Pathway. Methods in Molecular Biology, vol 2701. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3373-1_11
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