LAUSR

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 (&ggr;‐H2AX). However, &ggr;‐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 &ggr;‐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) &ggr;‐H2AX. Using optimized technical conditions, we found that &ggr;‐H2AX‐ub1 was a predominant form of &ggr;‐H2AX in three primary human cell lines containing mechanistically distinct types of DSBs. Replication stress‐associated DSBs also triggered extensive formation of &ggr;‐H2AX‐ub1. For DSBs induced by oxidative damage or topoisomerase II, both &ggr;‐H2AX and &ggr;‐H2AX‐ub1 showed dose‐dependent increases whereas &ggr;‐H2AX‐ub2 plateaued at low levels of breaks. Despite abundance of &ggr;‐H2AX, &ggr;‐H2AX‐ub1,2 formation was blocked in apoptosis, which was associated with proteolytic cleavage of RNF168. Chromatin damage also caused only the production of &ggr;‐H2AX but not its ub1,2 forms. Our results revealed a major contribution of ubiquitinated forms to the overall &ggr;‐H2AX response and demonstrated the specificity of monoubiquitinated &ggr;‐H2AX as a biodosimeter of non‐apoptotic DSBs. HIGHLIGHTSOptimized conditions for detection of monoubiquitinated &ggr;‐H2AX (&ggr;‐H2AX‐ub1)&ggr;‐H2AX‐ub1 is a main form in normal cells with DNA double‐strand breaks (DSBs)&ggr;‐H2AX‐ub1 is a dose‐dependent biomarker of DSBs arising as DNA damageUnlike &ggr;‐H2AX, &ggr;‐H2AX‐ub1 was absent in apoptotic cells or after chromatin damage