Abstract
DNA double-strand breaks (DSBs) are the most deleterious type of DNA damage and a cause of genetic instability as they can lead to mutations, genome rearrangements, or loss of genetic material when not properly repaired. Eukaryotes from budding yeast to mammalian cells respond to the formation of DSBs with the immediate phosphorylation of a histone H2A isoform. The modified histone, phosphorylated in serine 139 in mammals (S129 in yeast), is named γ-H2AX. Detection of DSBs is of high relevance in research on DNA repair, aging, tumorigenesis, and cancer drug development, given the tight association of DSBs with different diseases and its potential to kill cells. DSB levels can be obtained by measuring levels of γ-H2AX in extracts of cell populations or by counting foci in individual nuclei. In this chapter some techniques to detect γ-H2AX are described.
Key words
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM (1998) DNA double-strand breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273(10):5858–5868
Rogakou EP, Boon C, Redon C, Bonner WM (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 146(5):905–916
Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM (2000) A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 10(15):886–895
Downs JA, Lowndes NF, Jackson SP (2000) A role for Saccharomyces cerevisiae histone H2A in DNA repair. Nature 408(6815):1001–1004
Figueroa-González G, Pérez-Plasencia C (2017) Strategies for the evaluation of DNA damage and repair mechanisms in cancer. Oncol Lett 13(6):3982–3988. https://doi.org/10.3892/ol.2017.6002
Ostling O, Johanson KJ (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123(1):291–298
Anderson D, Dhawan A, Laubenthal J (2013) The comet assay in human biomonitoring. Methods Mol Biol 1044:347–362. https://doi.org/10.1007/978-1-62703-529-3_18
Kumari S, Rastogi R, Singh K, Singh S, Sinha R (2008) DNA damage: detection strategies. EXCLI J 7:44–62
Hu J, Meyers RM, Dong J, Panchakshari RA, Alt FW, Frock RL (2016) Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. Nat Protoc 11(5):853–871. https://doi.org/10.1038/nprot.2016.043
Levsky JM, Singer RH (2003) Fluorescence in situ hybridization: past, present and future. J Cell Sci 116(Pt 14):2833–2838
Jones JM, Gellert M, Yang W (2001) A Ku bridge over broken DNA. Structure 9(10):881–884
Bonner WM, Redon CE, Dickey JS, Nakamura AJ, Sedelnikova OA, Sollier S, Pommier Y (2008) Gamma H2AX and cancer. Nat Rev Cancer 8:957–967
Domínguez-Sánchez M, Barroso S, Gómez-González B, Luna R, Aguilera A (2011) Genome instability and transcription elongation impairment in human cells depleted of THO/TREX. PLoS Genet 7(12):e1002386. https://doi.org/10.1371/journal.pgen.1002386
Bhatia V, Barroso SI, García-Rubio ML, Tumini E, Herrera-Moyano E, Aguilera A (2014) BRCA2 prevents R-loop accumulation and associates with TREX-2 mRNA export factor PCID2. Nature 511(7509):362–365. https://doi.org/10.1038/nature13374
Acknowledgments
AA’s lab is funded by the Spanish Ministry of Economy and Competitiveness, Junta de Andalucía, European Research Council, and the European Union (FEDER).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Barroso, S.I., Aguilera, A. (2021). Detection of DNA Double-Strand Breaks by γ-H2AX Immunodetection. In: Aguilera, A., Carreira, A. (eds) Homologous Recombination. Methods in Molecular Biology, vol 2153. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0644-5_1
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0644-5_1
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0643-8
Online ISBN: 978-1-0716-0644-5
eBook Packages: Springer Protocols