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

Authors: Luczak, Michal W; Zhitkovich, Anatoly

Published In Toxicol Appl Pharmacol, (2018 09 15)

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, γ-H2AX-ub1,2 formation was blocked in apoptosis, which was associated with proteolytic cleavage of RNF168. Chromatin damage also caused only the production of γ-H2AX but not its ub1,2 forms. Our results revealed a major contribution of ubiquitinated forms to the overall γ-H2AX response and demonstrated the specificity of monoubiquitinated γ-H2AX as a biodosimeter of non-apoptotic DSBs.

PubMed ID: 30006243 Exiting the NIEHS site

MeSH Terms: Apoptosis/drug effects; Biomarkers/analysis*; Cell Line; Chromatin/drug effects; DNA Breaks, Double-Stranded/drug effects*; DNA Replication/drug effects; DNA Topoisomerases, Type II/metabolism; Dose-Response Relationship, Drug; Gene Knockdown Techniques; Heat-Shock Response; Histones/genetics; Histones/metabolism*; Humans; Oxidative Stress/drug effects; Stem Cells; Ubiquitination/drug effects*

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