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. Overall design: UV-induced CPD lesions are mapped using a high throughput method named CPD-seq. CPD-seq data generated in WT, RAD26 deletion, and Rad26 ATPase-dead yeast strains were analyzed to study the genome-wide role of Rad26 in TC-NER.

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）停滞信号，清除转录基因区域内的DNA损伤位点。TC-NER的激活依赖于特异性因子，例如人类科凯恩综合征B组（Cockayne syndrome group B, CSB）蛋白，或是其酵母同源蛋白Rad26。CSB基因突变与重症神经系统疾病科凯恩综合征密切相关。然而，目前学界对CSB/Rad26在TC-NER中的全基因组功能，尤其是在染色质组织背景下的作用机制，尚未完全阐明。

本研究借助单核苷酸分辨率的紫外线损伤定位数据，探究了Rad26在TC-NER中的全基因组功能。结果显示，Rad26对于首个（+1）核小体下游的转录区域内的TC-NER至关重要；但在+1核小体区域中，Rad26基本非必需。进一步研究发现，+1核小体区域内不依赖Rad26的TC-NER活性，与转录起始/修复因子TFIIH的高占据水平显著相关。在+1核小体下游区域，当Rad26功能异常时，TFIIH占据水平较低、转录延伸因子Spt4/Spt5占据水平较高的组合状态，会抑制TC-NER活性。敲除SPT4基因可显著恢复rad26缺失突变体中下游核小体区域的TC-NER活性。

综上，上述结果表明，TC-NER对Rad26的需求受TFIIH与Spt4/Spt5的分布模式调控，Rad26主要在下游核小体区域发挥功能，解除Spt4/Spt5对TC-NER的抑制作用。

实验设计概述：本研究借助名为CPD-seq的高通量检测方法，对紫外线诱导的环丁烷嘧啶二聚体（cyclobutane pyrimidine dimer, CPD）损伤位点进行定位。通过分析野生型、RAD26基因敲除型以及Rad26 ATP酶失活型酵母菌株的CPD-seq数据，解析Rad26在TC-NER中的全基因组功能。