Keogh Michael-Christopher, Kim Jung-Ae, Downey Michael, Fillingham Jeffrey, Chowdhury Dipanjan, Harrison Jacob C, Onishi Megumi, Datta Nira, Galicia Sarah, Emili Andrew, Lieberman Judy, Shen Xuetong, Buratowski Stephen, Haber James E, Durocher Daniel, Greenblatt Jack F, Krogan Nevan J
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Nature. 2006 Jan 26;439(7075):497-501. doi: 10.1038/nature04384. Epub 2005 Nov 20.
One of the earliest marks of a double-strand break (DSB) in eukaryotes is serine phosphorylation of the histone variant H2AX at the carboxy-terminal SQE motif to create gammaH2AX-containing nucleosomes. Budding-yeast histone H2A is phosphorylated in a similar manner by the checkpoint kinases Tel1 and Mec1 (ref. 2; orthologous to mammalian ATM and ATR, respectively) over a 50-kilobase region surrounding the DSB. This modification is important for recruiting numerous DSB-recognition and repair factors to the break site, including DNA damage checkpoint proteins, chromatin remodellers and cohesins. Multiple mechanisms for eliminating gammaH2AX as DNA repair completes are possible, including removal by histone exchange followed potentially by degradation, or, alternatively, dephosphorylation. Here we describe a three-protein complex (HTP-C, for histone H2A phosphatase complex) containing the phosphatase Pph3 that regulates the phosphorylation status of gammaH2AX in vivo and efficiently dephosphorylates gammaH2AX in vitro. gammaH2AX is lost from chromatin surrounding a DSB independently of the HTP-C, indicating that the phosphatase targets gammaH2AX after its displacement from DNA. The dephosphorylation of gammaH2AX by the HTP-C is necessary for efficient recovery from the DNA damage checkpoint.
真核生物中双链断裂（DSB）最早的标志之一是组蛋白变体H2AX在羧基末端SQE基序处发生丝氨酸磷酸化，从而形成含γH2AX的核小体。在出芽酵母中，组蛋白H2A在围绕DSB的50千碱基区域内，由检查点激酶Tel1和Mec1（分别与哺乳动物的ATM和ATR直系同源）以类似方式磷酸化。这种修饰对于将众多DSB识别和修复因子招募到断裂位点很重要，这些因子包括DNA损伤检查点蛋白、染色质重塑因子和黏连蛋白。随着DNA修复完成，有多种消除γH2AX的机制，包括通过组蛋白交换随后可能降解来去除，或者通过去磷酸化。在这里，我们描述了一种包含磷酸酶Pph3的三蛋白复合物（HTP-C，即组蛋白H2A磷酸酶复合物），它在体内调节γH2AX的磷酸化状态，并在体外有效地使γH2AX去磷酸化。γH2AX从围绕DSB的染色质中消失，这一过程与HTP-C无关，表明该磷酸酶在γH2AX从DNA上移位后靶向它。HTP-C对γH2AX的去磷酸化对于从DNA损伤检查点有效恢复是必要的。
