Abstract 1754: Monoubiquitinated γ-H2AX - a more specific biomarker of DNA double-strand breaks

Abstract

Abstract DNA double-strand brakes (DSBs) are a highly toxic form of DNA damage originating from many endogenous and exogenous sources. Cells employ specialized mechanisms for rapid detection and repair of these lesions. Histone H2AX phosphorylated at Ser139 (known as γ-H2AX) acts as a central platform for recruitment of other components of DSB repair. γ-H2AX assessment is currently the most widely used approach for quantitation of DSBs. However, γ-H2AX can be also induced by certain nongenotoxic stressors and during apoptosis, which can lead to false positive observations, especially in prolonged exposures. γ-H2AX undergoes RNF168-mediated K13/K15 monoubiquitination which is necessary for the recruitment of the downstream DSB repair factors. This ubiquitination event is rarely analyzed despite its functional importance. We examined formation and detection of γ-H2AX and its mono- (ub1) and diubiquitinated (ub2) forms in several human cell lines in response to mechanistically distinct inducers of DSBs and false-positive DSBs stressors. We found that γ-H2AX-ub forms were poorly detected using some common blotting procedures and antibodies. Under optimized conditions, γ-H2AX-ub1 was the predominant form accounting up to 80-90% of total γ-H2AX in primary human cells and its formation was strictly associated with the presence of DSBs. γ-H2AX and γ-H2AX-ub1 showed similar dose dependence and disappearance kinetics whereas H2AX-ub2 form was a more stable and dose-independent product. Unlike γ-H2AX, its ubiquitinated forms were not observed in the absence of DSBs (heat-shock or during apoptotic DNA cleavage). Apoptotic cells showed cleavage of the γ-H2AX-targeting E3 ubiquitin ligase RNF168. Our findings demonstrate that the ub1 form is a major fraction in the overall formation of γ-H2AX and its quantitation offers advantage in specificity for nonapoptotic DSBs. Citation Format: Michal W. Luczak, Anatoly Zhitkovich. Monoubiquitinated γ-H2AX - a more specific biomarker of DNA double-strand breaks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1754.