Sin Yooksil, Tanaka Kiyoji, Saijo Masafumi
From the Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, Suita, Osaka 565-0871, Japan.
From the Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, Suita, Osaka 565-0871, Japan
J Biol Chem. 2016 Jan 15;291(3):1387-97. doi: 10.1074/jbc.M115.683235. Epub 2015 Nov 30.
Cockayne syndrome (CS) is a recessive disorder that results in deficiencies in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair, and cells from CS patients exhibit hypersensitivity to UV light. CS group B protein (CSB), which is the gene product of one of the genes responsible for CS, belongs to the SWI2/SNF2 DNA-dependent ATPase family and has an ATPase domain and an ubiquitin-binding domain (UBD) in the central region and the C-terminal region, respectively. The C-terminal region containing the UBD is essential for the functions of CSB. In this study, we generated several CSB deletion mutants and analyzed the functions of the C-terminal region of CSB in TC-NER. Not only the UBD but also the C-terminal 30-amino acid residues were required for UV light resistance and TC-NER. This region was needed for the interaction of CSB with RNA polymerase II, the translocation of CS group A protein to the nuclear matrix, and the association of CSB with chromatin after UV irradiation. CSB was modified by small ubiquitin-like modifier 2/3 in a UV light-dependent manner. This modification was abolished in a CSB mutant lacking the C-terminal 30 amino acid residues. However, the substitution of lysine residues in this region with arginine did not affect SUMOylation or TC-NER. By contrast, substitution of a lysine residue in the N-terminal region with arginine decreased SUMOylation and resulted in cells with defects in TC-NER. These results indicate that both the most C-terminal region and SUMOylation are important for the functions of CSB in TC-NER.
科凯恩综合征（CS）是一种隐性疾病，会导致转录偶联核苷酸切除修复（TC-NER）功能缺陷，TC-NER是核苷酸切除修复的一个子途径，CS患者的细胞对紫外线敏感。CS B组蛋白（CSB）是导致CS的其中一个基因的产物，属于SWI2/SNF2 DNA依赖性ATP酶家族，在中央区域和C末端区域分别具有一个ATP酶结构域和一个泛素结合结构域（UBD）。包含UBD的C末端区域对CSB的功能至关重要。在本研究中，我们构建了多个CSB缺失突变体，并分析了CSB C末端区域在TC-NER中的功能。不仅UBD，而且C末端的30个氨基酸残基对于抗紫外线和TC-NER都是必需的。该区域对于CSB与RNA聚合酶II的相互作用、CS A组蛋白向核基质的转运以及紫外线照射后CSB与染色质的结合都是必需的。CSB以紫外线依赖性方式被小泛素样修饰物2/3修饰。在缺乏C末端30个氨基酸残基的CSB突变体中，这种修饰被消除。然而，该区域中赖氨酸残基被精氨酸取代并不影响SUMO化或TC-NER。相比之下，N末端区域中的一个赖氨酸残基被精氨酸取代会降低SUMO化，并导致细胞在TC-NER方面存在缺陷。这些结果表明，最末端区域和SUMO化对于CSB在TC-NER中的功能都很重要。
