Kleiner Ralph E, Verma Priyanka, Molloy Kelly R, Chait Brian T, Kapoor Tarun M
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, New York, USA.
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York, USA..
Nat Chem Biol. 2015 Oct;11(10):807-14. doi: 10.1038/nchembio.1908. Epub 2015 Sep 7.
DNA double-strand break repair involves phosphorylation of histone variant H2AX ('γH2AX'), which accumulates in foci at sites of DNA damage. In current models, the recruitment of multiple DNA repair proteins to γH2AX foci depends mainly on recognition of this 'mark' by a single protein, MDC1. However, DNA repair proteins accumulate at γH2AX sites without MDC1, suggesting that other 'readers' of this mark exist. Here, we use a quantitative chemical proteomics approach to profile direct, phospho-selective γH2AX binders in native proteomes. We identify γH2AX binders, including the DNA repair mediator 53BP1, which we show recognizes γH2AX through its BRCT domains. Furthermore, we investigate the targeting of wild-type 53BP1, or a mutant form deficient in γH2AX binding, to chromosomal breaks resulting from endogenous and exogenous DNA damage. Our results show how direct recognition of γH2AX modulates protein localization at DNA damage sites, and suggest how specific chromatin mark-reader interactions contribute to essential mechanisms ensuring genome stability.
DNA双链断裂修复涉及组蛋白变体H2AX（“γH2AX”）的磷酸化，γH2AX会在DNA损伤位点处聚集形成病灶。在当前模型中，多种DNA修复蛋白募集到γH2AX病灶主要依赖于单一蛋白MDC1对这种“标记”的识别。然而，DNA修复蛋白在没有MDC1的情况下也会在γH2AX位点聚集，这表明存在其他识别这种标记的“读取器”。在此，我们使用定量化学蛋白质组学方法对天然蛋白质组中直接的、磷酸化选择性的γH2AX结合蛋白进行分析。我们鉴定出了γH2AX结合蛋白，包括DNA修复介质53BP1，我们发现它通过其BRCT结构域识别γH2AX。此外，我们研究了野生型53BP1或缺乏γH2AX结合能力的突变形式靶向内源性和外源性DNA损伤导致的染色体断裂的情况。我们的结果展示了γH2AX的直接识别如何调节蛋白质在DNA损伤位点的定位，并揭示了特定染色质标记读取器相互作用如何促成确保基因组稳定性的基本机制。
