CPD-seq mapping of transcription-coupled DNA repair in yeast

Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that responds to RNA polymerase (RNAP) stalling and removes DNA lesions from transcribed genes. Activation of TC-NER requires specific factors, such as human Cockayne syndrome group B (CSB) protein or its yeast homolog Rad26. Mutations in CSB are associated with the severe neurological disorder Cockayne syndrome. However, the genome-wide role of CSB/Rad26 in TC-NER, particularly in the context of chromatin organization, is not fully understood. Here we used single-nucleotide resolution UV damage mapping data to investigate the genome-wide function of Rad26 in TC-NER. Our data shows that Rad26 is critical for TC-NER in transcribed regions downstream of the first (+1) nucleosome; however, Rad26 is largely dispensable for TC-NER in the +1 nucleosome. We further show that the Rad26-independent TC-NER in the +1 nucleosome is correlated with high occupancy of the transcription initiation/repair factor TFIIH. Downstream of the +1 nucleosome, the combination of low TFIIH occupancy and high occupancy of the transcription elongation factor Spt4/Spt5 suppresses TC-NER when Rad26 is dysfunctional. Deletion of SPT4 significantly restores TC-NER in the downstream nucleosomes in a rad26∆ mutant. Collectively, these data indicate that the requirement for Rad26 in TC-NER is modulated by the distribution of TFIIH and Spt4/Spt5, and Rad26 mainly functions in the downstream nucleosomes to remove TC-NER suppression by Spt4/Spt5.转录偶联核苷酸切除修复（Transcription-coupled nucleotide excision repair, TC-NER）是一类重要的DNA修复机制，可响应RNA聚合酶（RNA polymerase, RNAP）的停滞，并从转录基因中清除DNA损伤。TC-NER的激活需要特定因子，例如人类科凯恩综合征B组蛋白（Cockayne syndrome group B, CSB）或其酵母同源物Rad26。CSB的突变与重症神经系统疾病科凯恩综合征密切相关。然而，CSB/Rad26在TC-NER中的全基因组作用，尤其是在染色质组织背景下，尚未得到完全阐明。本研究利用单核苷酸分辨率UV损伤图谱数据，探究了Rad26在TC-NER中的全基因组功能。研究数据表明，Rad26对首个（+1）核小体下游转录区域的TC-NER至关重要；但在+1核小体区域的TC-NER中，Rad26基本非必需。我们进一步发现，+1核小体中不依赖Rad26的TC-NER，与转录起始/修复因子TFIIH（Transcription Factor II H）的高结合占据率相关。在+1核小体下游区域，低TFIIH结合占据率与转录延伸因子Spt4/Spt5的高结合占据率共同作用，会在Rad26功能异常时抑制TC-NER。敲除SPT4可显著恢复rad26Δ突变体下游核小体的TC-NER活性。综上，本研究数据表明，TC-NER对Rad26的需求受TFIIH与Spt4/Spt5的分布模式调控，且Rad26主要在下游核小体中发挥功能，以解除Spt4/Spt5对TC-NER的抑制。