Monoubiquitinated γ-H2AX: Abundant product and specific biomarker for non-apoptotic DNA double-strand breaks

Toxicology and Applied Pharmacology
Michal W Luczak, Anatoly Zhitkovich

Abstract

DNA double-strand breaks (DSBs) are a highly toxic form of DNA damage produced by a number of carcinogens, drugs, and metabolic abnormalities. Involvement of DSBs in many pathologies has led to frequent measurements of these lesions, primarily via biodosimetry of S139-phosphorylated histone H2AX (γ-H2AX). However, γ-H2AX is also induced by some non-DSB conditions and abundantly formed in apoptosis, raising concerns about the overestimation of potential genotoxic agents and accuracy of DSB assessments. DSB-triggered γ-H2AX undergoes RNF168-mediated K13/K15 monoubiquitination, which is rarely analyzed in DSB/genotoxicity studies. Here we identified critical methodological factors that are necessary for the efficient detection of mono- (ub1) and diubiquitinated (ub2) γ-H2AX. Using optimized technical conditions, we found that γ-H2AX-ub1 was a predominant form of γ-H2AX in three primary human cell lines containing mechanistically distinct types of DSBs. Replication stress-associated DSBs also triggered extensive formation of γ-H2AX-ub1. For DSBs induced by oxidative damage or topoisomerase II, both γ-H2AX and γ-H2AX-ub1 showed dose-dependent increases whereas γ-H2AX-ub2 plateaued at low levels of breaks. Despite abundance of γ-H2AX...Continue Reading

Citations

Jul 11, 2019·Archives of Toxicology·B KoppMarc Audebert
Aug 8, 2019·Toxicological Sciences : an Official Journal of the Society of Toxicology·Michal W LuczakAnatoly Zhitkovich
Jun 27, 2019·Current Protocols in Cytometry·Hong ZhaoZbigniew Darzynkiewicz
Aug 18, 2020·Molecular Cancer Research : MCR·Shruthi SriramkumarHeather M O'Hagan
Feb 26, 2021·Toxicology in Vitro : an International Journal Published in Association with BIBRA·Kevin HogeveenValérie Fessard
Mar 16, 2021·Dose-response : a Publication of International Hormesis Society·Yue HuangNaiyao Chen
Oct 27, 2018·Chemical Research in Toxicology·Casey Krawic, Anatoly Zhitkovich

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