Rad26, the Transcription-Coupled Repair Factor in Yeast, Is Required for Removal of Stalled RNA Polymerase-II following UV Irradiation

Transcription coupled nucleotide excision repair (TCR) is a major pathway responsible for removal of helix distorting DNA lesions from transcriptionally active regions of the genome. Rad26, a key factor of the TCR pathway, is known to play a role during early steps of TCR. Here, we show that Rad26-mediated TCR is not absolutely dependent on active transcription elongation in budding yeast. As per our results, RAD26-deleted cells show enhanced UV sensitivity compared to wild type cells under conditions where transcription elongation is inhibited. The increased UV sensitivity observed in RAD26-deleted cells, however, is not due to reduced expression of the major NER-responsive genes. Interestingly, transcription of the constitutively expressed RPB2 gene is adversely affected in RAD26-deleted cells during UV-induced DNA damage repair. In consonance, chromatin immunoprecipitation analysis showed that unlike in wild type, in RAD26-deleted cells no significant reduction in RNA polymerase II occupancy occurs during nucleotide excision repair in the transcriptionally active loci like, RPB2, PYK1 and RPL2B. These results collectively indicate that removal of RNAPII during DNA damage repair following UV irradiation is dependent on Rad26.

转录偶联核苷酸切除修复（Transcription coupled nucleotide excision repair, TCR）是负责从基因组转录活跃区域移除螺旋扭曲型DNA损伤的核心通路。Rad26作为TCR通路的关键因子，已知在TCR的早期阶段发挥功能。本研究证实，在酿酒酵母中，Rad26介导的TCR并非绝对依赖于活跃的转录延伸过程。实验结果显示，在转录延伸受到抑制的条件下，RAD26缺失细胞相较于野生型细胞，其紫外线敏感性显著升高。值得注意的是，RAD26缺失细胞中观测到的紫外线敏感性增强，并非由主要的核苷酸切除修复（nucleotide excision repair, NER）响应基因的表达水平下调所导致。有趣的是，在紫外线诱导的DNA损伤修复过程中，组成型表达的RPB2基因的转录在RAD26缺失细胞中受到显著负面影响。与此结论一致的是，染色质免疫沉淀（chromatin immunoprecipitation, ChIP）分析结果表明：与野生型细胞相比，RAD26缺失细胞在转录活跃基因座（如RPB2、PYK1及RPL2B）进行核苷酸切除修复时，RNA聚合酶II的结合丰度未出现显著下降。上述结果共同表明，紫外线照射后DNA损伤修复过程中RNA聚合酶II的移除依赖于Rad26。