Institute for Quantitative Biosciences, The University of Tokyo, Tokyo 113-0032, Japan.
Synchrotron Radiation Research Organization, The University of Tokyo, Tokyo 113-0032, Japan.
Nucleic Acids Res. 2019 Apr 23;47(7):3784-3794. doi: 10.1093/nar/gkz081.
Cockayne syndrome group B (CSB, also known as ERCC6) protein is involved in many DNA repair processes and essential for transcription-coupled repair (TCR). The central region of CSB has the helicase motif, whereas the C-terminal region contains important regulatory elements for repair of UV- and oxidative stress-induced damages and double-strand breaks (DSBs). A previous study suggested that a small part (∼30 residues) within this region was responsible for binding to ubiquitin (Ub). Here, we show that the Ub-binding of CSB requires a larger part of CSB, which was previously identified as a winged-helix domain (WHD) and is involved in the recruitment of CSB to DSBs. We also present the crystal structure of CSB WHD in complex with Ub. CSB WHD folds as a single globular domain, defining a class of Ub-binding domains (UBDs) different from 23 UBD classes identified so far. The second α-helix and C-terminal extremity of CSB WHD interact with Ub. Together with structure-guided mutational analysis, we identified the residues critical for the binding to Ub. CSB mutants defective in the Ub binding reduced repair of UV-induced damage. This study supports the notion that DSB repair and TCR may be associated with the Ub-binding of CSB.
Cockayne 综合征 B 组（CSB，也称为 ERCC6）蛋白参与多种 DNA 修复过程，是转录偶联修复（TCR）所必需的。CSB 的中心区域具有解旋酶基序，而 C 末端区域包含修复 UV 和氧化应激诱导的损伤和双链断裂（DSB）的重要调节元件。先前的研究表明，该区域的一小部分（约 30 个残基）负责与泛素（Ub）结合。在这里，我们表明 CSB 的 Ub 结合需要 CSB 的更大一部分，这部分先前被鉴定为翼状螺旋结构域（WHD），并参与 CSB 招募到 DSB 中。我们还展示了 CSB WHD 与 Ub 复合物的晶体结构。CSB WHD 折叠成一个单一的球形结构域，定义了一类不同于迄今为止鉴定的 23 种 Ub 结合结构域（UBD）的 Ub 结合结构域。CSB WHD 的第二个α-螺旋和 C 末端与 Ub 相互作用。通过结构引导的突变分析，我们确定了与 Ub 结合的关键残基。在 Ub 结合中存在缺陷的 CSB 突变体减少了对 UV 诱导损伤的修复。这项研究支持这样一种观点，即 DSB 修复和 TCR 可能与 CSB 的 Ub 结合有关。
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