Division of Biochemistry and Center for Biomedical Genetics, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Cell. 2012 Sep 14;150(6):1182-95. doi: 10.1016/j.cell.2012.08.005.
Ubiquitin-dependent signaling during the DNA damage response (DDR) to double-strand breaks (DSBs) is initiated by two E3 ligases, RNF8 and RNF168, targeting histone H2A and H2AX. RNF8 is the first ligase recruited to the damage site, and RNF168 follows RNF8-dependent ubiquitination. This suggests that RNF8 initiates H2A/H2AX ubiquitination with K63-linked ubiquitin chains and RNF168 extends them. Here, we show that RNF8 is inactive toward nucleosomal H2A, whereas RNF168 catalyzes the monoubiquitination of the histones specifically on K13-15. Structure-based mutagenesis of RNF8 and RNF168 RING domains shows that a charged residue determines whether nucleosomal proteins are recognized. We find that K63 ubiquitin chains are conjugated to RNF168-dependent H2A/H2AX monoubiquitination at K13-15 and not on K118-119. Using a mutant of RNF168 unable to target histones but still catalyzing ubiquitin chains at DSBs, we show that ubiquitin chains per se are insufficient for signaling, but RNF168 target ubiquitination is required for DDR.
在双链断裂 (DSB) 的 DNA 损伤反应 (DDR) 过程中，泛素依赖性信号是由两种 E3 连接酶 RNF8 和 RNF168 启动的，它们靶向组蛋白 H2A 和 H2AX。RNF8 是第一个被招募到损伤部位的连接酶，而 RNF168 则依赖于 RNF8 的泛素化。这表明 RNF8 以 K63 连接的泛素链起始 H2A/H2AX 的泛素化，而 RNF168 则延伸它们。在这里，我们表明 RNF8 对核小体 H2A 无活性，而 RNF168 则特异性地在 K13-15 上催化组蛋白的单泛素化。RNF8 和 RNF168 RING 结构域的基于结构的突变显示，一个带电残基决定了核小体蛋白是否被识别。我们发现 K63 泛素链被连接到 RNF168 依赖性的 H2A/H2AX 单泛素化上，而不是在 K118-119 上。使用一种不能靶向组蛋白但仍能在 DSB 处催化泛素链的 RNF168 突变体，我们表明泛素链本身不足以进行信号传递，但 DDR 需要 RNF168 靶向的泛素化。
