Abstract
Phosphorylation of histone H2AX on Ser 139 is a sensitive reporter of DNA damage, particularly if the damage involves induction of DNA double-strand breaks (DSBs). Phosphorylated H2AX has been named γH2AX and its presence in the nucleus can be detected immunocytochemically. Multiparameter analysis of γH2AX immunofluorescence by flow or laser-scanning cytometry allows one to measure extent of DNA damage in individual cells and to correlate it with their position in the cell cycle and induction of apoptosis. This chapter presents the protocols and outlines applications of multiparameter cytometry in analysis of H2AX phosphorylation as a reporter of the presence of DSBs.
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-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 273, 5858–5868.
Sedelnikova OA, Rogakou EP, Panuytin IG, Bonner W. (2002) Quantitative detection of 125IUdr-induced DNA double-strand breaks with γ-H2AX antibody. Radiation Res. 158, 486–492.
West MH, Bonner WM. (1980) Histone 2A, a heteromorphous family of eight protein species. Biochemistry. 19, 3238–3245.
Thatcher TH, Gorovsky MA. (1994) Phylogenetic analysis of the core histones H2A, H2B, H3 and H4. Nucleic Acids Res. 22, 174–179.
Pehrson JR, Fuji RN. (1998) Evolutionary conservation of histone macro-H2A subtypes and domains. Nuclei Acids Res. 26, 2837–2842.
Burma S, Chen BP, Murphy M, Kurimasa A, Chen DJ. (2001) ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem. 276, 42462–42467.
Anderson L, Henderson C, Adachi Y. (2001) Phosphorylation and rapid relocalization of 53BP1 to nuclear foci upon DNA damage. Mol Cell Biol. 21, 1719–1729.
Furuta T, Takemura H, Liao-Z-Y, Aune GJ, Redon C, Sedelnikova OA, Pilch DR, Rogakou EP, Celeste A, Chen HT, Nussenzweig A, Aladjem MI, Bonner WM, Pommier Y. (2003) Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induces by mammalian topoisomerase I cleavage complexes. J Biol Chem. 278, 20303–20312.
Park EJ, Chan DW, Park JH, Oettinger MA, Kwon J. (2003) DNA-PK is activated by nucleosomes and phosphorylated H2AX within the nucleosomes in an acetylation-dependent manner. Nuclei Acids Res. 31, 6819–6827.
Wang H, Wang M, Wang H, Bocker W, Iliakis G. (2005) Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA-PK in human cells exposed to ionizing radiation and treated with kinase inhibitors. J Cell Physiol. 202, 492–502.
Rogakou EP, Boon C, Redon, C, Bonner WM. (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol. 146, 905–916.
Banath JP, Olive PL. (2003) Expression of phosphorylated histone H2AX as a surrogate of cell killing by drugs that create DNA double-strand breaks. Cancer Res. 63, 4347–4350.
Huang X, Okafuji M, Traganos F, Luther E, Holden E, Darzynkiewicz Z. (2004) Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors Topotecan and Mitoxantrone and by the DNA cross-linking agent Cisplatin. Cytometry. 58A, 99–110.
Paull TT, Rogakou EP, Yamazaki V, Kirchgesser 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, 886–895.
Bassing CH, Alt FW. (2004) H2AX may function as an anchor to hold broken chromosomal DNA ends in close proximity. Cell Cycle. 3, 149–153.
Tanaka T, Kurose A, Halicka HD, Traganos F, Darzynkiewicz Z. (2006) 2-Deoxy-D-glucose reduces the level of constitutive activation of ATM and phosphorylation of histone H2AX. Cell Cycle. 5, 878–882.
Tanaka T, Kurose A, Halicka HD, Huang X, Traganos F, Darzynkiewicz Z. (2006) Nitrogen oxide-releasing aspirin induces histone H2AX phosphorylation, ATM activation and apoptosis preferentially in S-phase cells. Cell Cycle. 5, 1669–1674.
Tanaka T, Halicka HD, Huang X, Traganos F, Darzynkiewicz Z. (2006) Constitutive histone H2AX phosphorylation and ATM activation, the reporters of DNA damage by endogenous oxidants. Cell Cycle. 5, 1940–1945.
Tanaka T, Huang X, Jorgensen E, Gietl E, Traganos F, Darzynkiewicz Z, Albino AP. (2007) ATM activation accompanies histone H2AX phosphorylation in A549 cells upon exposure to tobacco smoke. BMC Cell Biol. 8, 26.
Tanaka T, Huang X, Halicka HD, Zhao H, Traganos F, Albino AP, Dai W, Darzynkiewicz Z. (2007) Cytometry of ATM activation and histone H2AX phosphorylation to estimate extent of DNA damage induced by exogenous agents. Cytometry 71A, 648–661.
Kurose A, Tanaka T, Huang X, Traganos F, Dai W, Darzynkiewicz Z (2006). Effects of hydroxyurea and aphidicolin on phosphorylation of ATM on Ser 1981 and histone H2AX on Ser 139 in relation to cell cycle phase and induction of apoptosis. Cytometry A 69A, 212–221.
Huang X, Kurose A, Tanaka T, Traganos F, Dai W, Darzynkiewicz Z (2006) Sequential phosphorylation of Ser-10 on histone H3 and Ser-139 on histone H2AX and ATM activation during premature chromosome condensation: Relationship to cell-cycle and apoptosis. Cytometry A 69A, 222–229.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Tanaka, T., Halicka, D., Traganos, F., Darzynkiewicz, Z. (2009). Cytometric Analysis of DNA Damage: Phosphorylation of Histone H2AX as a Marker of DNA Double-Strand Breaks (DSBs). In: Chellappan, S. (eds) Chromatin Protocols. Methods in Molecular Biology, vol 523. Humana Press. https://doi.org/10.1007/978-1-59745-190-1_11
Download citation
DOI: https://doi.org/10.1007/978-1-59745-190-1_11
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-873-7
Online ISBN: 978-1-59745-190-1
eBook Packages: Springer Protocols