Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that removes RNA polymerase (RNAP)-stalling DNA damage from the transcribed strand (TS) of active genes. TC-NER deficiency in humans is associated with the severe neurological disorder Cockayne syndrome. Initiation of TC-NER is mediated by specific factors such as the human Cockayne syndrome group B (CSB) protein or its yeast homolog Rad26. However, the genome-wide role of CSB/Rad26 in TC-NER, particularly in the context of the chromatin organization, is unclear. Here, we used single-nucleotide resolution UV damage mapping data to show that Rad26 and its ATPase activity is critical for TC-NER downstream of the first (+1) nucleosome in gene coding regions. However, TC-NER on the transcription start site (TSS)-proximal half of the +1 nucleosome is largely independent of Rad26, likely due to high occupancy of the transcription initiation/repair factor TFIIH in this nucleosome. Downstream of the +1 nucleosome, the combination of low TFIIH occupancy and high occupancy of the transcription elongation factor Spt4/Spt5 suppresses TC-NER in Rad26-deficient cells. We show that deletion of SPT4 significantly restores TC-NER across the genome in a rad26∆ mutant, particularly in the downstream nucleosomes. These data demonstrate that the requirement for Rad26 in TC-NER is modulated by the distribution of TFIIH and Spt4/Spt5 in transcribed chromatin and Rad26 mainly functions downstream of the +1 nucleosome to remove TC-NER suppression by Spt4/Spt5.

转录偶联核苷酸切除修复（TC-NER）是一种重要的DNA修复机制，可从活性基因的转录链（TS）中消除RNA聚合酶（RNAP）造成的DNA损伤。人体中的TC-NER缺乏与严重的神经系统疾病Cockayne综合征有关。TC-NER的启动由特定因素介导，例如人类Cockayne综合征B组（CSB）蛋白或其酵母同源物Rad26。但是，尚不清楚CSB / Rad26在TC-NER中的全基因组作用，尤其是在染色质组织的情况下。在这里，我们使用单核苷酸分辨率的UV损伤定位数据来显示Rad26及其ATPase活性对于基因编码区域中第一个（+1）核小体下游的TC-NER至关重要。然而，转录起始位点（TSS）-+ 1核小体的近半部分上的TC-NER在很大程度上不依赖于Rad26，这可能是由于该核小体中转录起始/修复因子TFIIH的占有率很高。+1核小体的下游，低的TFIIH占有率和高的转录延伸因子Spt4 / Spt5占有率的组合抑制了Rad26缺陷细胞中的TC-NER。我们显示删除SPT4在rad26Δ突变体中，尤其是在下游核小体中，可在整个基因组中显着恢复TC-NER 。这些数据表明，TF-IIH和Spt4 / Spt5在转录的染色质中的分布调节了对TC-NER中Rad26的需求，Rad26主要在+1核小体的下游起作用，以消除Spt4 / Spt5对TC-NER的抑制作用。