School of Molecular Biosciences, Washington State University, Pullman, WA, USA.
Division of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA.
Nat Commun. 2024 Jul 23;15(1):6223. doi: 10.1038/s41467-024-50539-y.
Transcription coupled-nucleotide excision repair (TC-NER) removes DNA lesions that block RNA polymerase II (Pol II) transcription. A key step in TC-NER is the recruitment of the TFIIH complex, which initiates DNA unwinding and damage verification; however, the mechanism by which TFIIH is recruited during TC-NER, particularly in yeast, remains unclear. Here, we show that the C-terminal domain (CTD) of elongation factor-1 (Elf1) plays a critical role in TC-NER in yeast by binding TFIIH. Analysis of genome-wide repair of UV-induced cyclobutane pyrimidine dimers (CPDs) using CPD-seq indicates that the Elf1 CTD in yeast is required for efficient TC-NER. We show that the Elf1 CTD binds to the pleckstrin homology (PH) domain of the p62 subunit of TFIIH in vitro, and identify a putative TFIIH-interaction region (TIR) in the Elf1 CTD that is important for PH binding and TC-NER. The Elf1 TIR shows functional, structural, and sequence similarities to a conserved TIR in the mammalian UV sensitivity syndrome A (UVSSA) protein, which recruits TFIIH during TC-NER in mammalian cells. These findings suggest that the Elf1 CTD acts as a functional counterpart to mammalian UVSSA in TC-NER by recruiting TFIIH in response to Pol II stalling at DNA lesions.
转录偶联核苷酸切除修复 (TC-NER) 可去除阻止 RNA 聚合酶 II (Pol II) 转录的 DNA 损伤。TC-NER 的一个关键步骤是募集 TFIIH 复合物，该复合物启动 DNA 解旋和损伤验证；然而，TFIIH 在 TC-NER 过程中的募集机制，特别是在酵母中，仍然不清楚。在这里，我们通过结合 TFIIH 表明伸长因子-1 (Elf1) 的 C 端结构域 (CTD) 在酵母中的 TC-NER 中发挥关键作用。使用 CPD-seq 分析紫外线诱导的环丁烷嘧啶二聚体 (CPD) 的全基因组修复表明，酵母中的 Elf1 CTD 是有效 TC-NER 所必需的。我们表明，Elf1 CTD 在体外与 TFIIH 的 p62 亚基的 pleckstrin 同源 (PH) 结构域结合，并鉴定出 Elf1 CTD 中的一个推定的 TFIIH 相互作用区域 (TIR)，该区域对于 PH 结合和 TC-NER 很重要。Elf1 TIR 显示出与哺乳动物紫外线敏感综合征 A (UVSSA) 蛋白中的保守 TIR 具有功能、结构和序列相似性，该蛋白在哺乳动物细胞中的 TC-NER 过程中募集 TFIIH。这些发现表明，Elf1 CTD 通过响应 Pol II 在 DNA 损伤处的停滞而募集 TFIIH，作为 TC-NER 中哺乳动物 UVSSA 的功能对应物。
