Luczak Michal W, Zhitkovich Anatoly
Department of Pathology and Laboratory Medicine, Brown University, Providence, RI 02912, USA.
Department of Pathology and Laboratory Medicine, Brown University, Providence, RI 02912, USA.
Toxicol Appl Pharmacol. 2018 Sep 15;355:238-246. doi: 10.1016/j.taap.2018.07.007. Epub 2018 Jul 10.
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- (ub) and diubiquitinated (ub) γ-H2AX. Using optimized technical conditions, we found that γ-H2AX-ub 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-ub. For DSBs induced by oxidative damage or topoisomerase II, both γ-H2AX and γ-H2AX-ub showed dose-dependent increases whereas γ-H2AX-ub plateaued at low levels of breaks. Despite abundance of γ-H2AX, γ-H2AX-ub 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 ub 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.
DNA双链断裂（DSB）是由多种致癌物、药物和代谢异常产生的一种高毒性DNA损伤形式。DSB参与多种病理过程，导致人们经常对这些损伤进行检测，主要是通过对S139磷酸化组蛋白H2AX（γ-H2AX）进行生物剂量测定。然而，γ-H2AX也可由一些非DSB条件诱导产生，并在细胞凋亡过程中大量形成，这引发了人们对潜在遗传毒性剂的高估以及DSB评估准确性的担忧。DSB引发的γ-H2AX会经历RNF168介导的K13/K15单泛素化，而这在DSB/遗传毒性研究中很少被分析。在这里，我们确定了高效检测单泛素化（ub）和双泛素化（ub）γ-H2AX所需的关键方法学因素。使用优化的技术条件，我们发现γ-H2AX-ub是三种含有机制不同类型DSB的原代人细胞系中γ-H2AX的主要形式。与复制应激相关的DSB也会引发γ-H2AX-ub的大量形成。对于由氧化损伤或拓扑异构酶II诱导的DSB，γ-H2AX和γ-H2AX-ub均呈剂量依赖性增加，而γ-H2AX-ub在低水平断裂时达到平台期。尽管γ-H2AX含量丰富，但γ-H2AX-ub的形成在细胞凋亡过程中被阻断，这与RNF168的蛋白水解切割有关。染色质损伤也仅导致γ-H2AX的产生，而不产生其泛素化形式。我们的结果揭示了泛素化形式对整体γ-H2AX反应的主要贡献，并证明了单泛素化γ-H2AX作为非凋亡DSB生物剂量计的特异性。
