Mechanism of the initial steps in transcription-coupled DNA repair (TCR)

对S.酿酒酵母中的TCR起始不同于哺乳动物细胞中的TCR起始。虽然Cockayne综合征组B基因的缺失严重抑制哺乳动物细胞中的TCR，但其酵母同源物Rad 26的缺失仅轻微损害TCR。遗传分析强烈提示酵母中有两种替代TCR子途径。第一个主导途径可能是由Pol II与Rad 26的相互作用启动的，并且依赖于非必需的Pol II亚基Rpb 4。第二TCR途径在不存在Rpb 4的情况下变得突出，并且依赖于另一个非必需Pol II亚基Rpb 9。Rpb 9介导的TCR途径的机制还不清楚。由于缺乏可能致死的RPB 4/RPB 9双缺失突变体，通过遗传手段对其进行的研究受到阻碍。对酵母中Rpb 9依赖性通路的分析可能为TCR期间Pol II相关事件提供重要见解。Rpb 9亚基在Pol II周边的位置表明其可能的功能是将NER因子募集到受损部位。Rpb 9参与多种转录相关功能，如转录起始（起始位点的选择），转录延伸，以及最近参与响应UV诱导的DNA损伤的rpb 1的泛素化和降解。该亚基还与参与转录延伸和组织修饰的大量因子（如TFIIS、TFIIE和佐贺）相互作用。这些因子中的哪一个在Rpb 9介导的TCR途径中充当Rad 26类似物仍有待鉴定。今年（2012年），该项目导致在Molecular Cell上发表的手稿展示了酵母Pol II通过CPD病变进行转录的机制。UV诱导的模板DNA链中的环丁烷嘧啶二聚体（CPD）通过Pol II停止转录延伸。如果核苷酸切除修复机制不能迅速去除CPD，停滞的Pol II会为DNA复制和随后的转录轮次造成障碍。在这里，我们提出的证据表明，Pol II有一个内在的能力translesion合成（TLS），使绕过CPD或不修复。转位合成依赖于触发环和桥螺旋，这两个灵活的区域的Pol II亚基Rpb 1参与底物结合，催化和转位。Rpb 1中的取代促进体外病变绕过增加体内UV抗性，而抑制病变绕过的取代减少UV照射后的细胞存活。这项工作揭示了在基因组DNA中未修复的CPD损伤积累后，跨损伤转录对细胞存活的重要性。

The mechanism of TCR initiation in S. cerevisiae is distinct from the TCR initiation in mammalian cells. While deletion of the Cockayne Syndrome Group B gene severely inhibits TCR in the mammalian cells, deletion of its yeast homologue Rad26 only slightly impairs the TCR. Genetic analyses strongly suggest two alternative TCR subpathways in yeast. The first, dominant pathway is probably initiated by Pol II interaction with Rad26, and is dependent on a non-essential Pol II subunit Rpb4. The second TCR pathway becomes prominent in the absence of Rpb4, and is dependent on another non-essential Pol II subunit Rpb9. The mechanism of the Rpb9-mediated TCR pathway is not well understood. Its investigation by genetic means has been hampered by the lack of the RPB4/RPB9 double deletion mutant, which is likely to be lethal. Analysis of the Rpb9-dependent pathway in yeast may provideimportant insights into the Pol II-related events during TCR. The location of the Rpb9 subunit on the perimeter of Pol II suggests its possible function in recruiting NER factors to the damaged site. Rpb9 is involved in multiple-transcription related functions such as transcription initiation (selection of the start site), transcription elongation, and recently in ubiquitination and degradation of rpb1 in response to UV-induced DNA damage. This subunit also interacts with a plethora of factors involved in transcription elongation and histonemodification (like TFIIS, TFIIE, and SAGA). Which of these factors act as a Rad26 analogue in the Rpb9-mediated TCR pathway remains to be identified.This year (2012) this project resulted in publication of the manuscript in Molecular Cell demonstrating the mechanism employed by the yeast Pol II for transcription through the CPD lesions. UV-induced cyclobutane pyrimidine dimers (CPDs) in the template DNA strand stall transcription elongation by Pol II. If the nucleotide excision repair machinery does not promptly remove the CPDs, stalled Pol II creates a roadblock for DNA replication and subsequent rounds of transcription. Here we present evidence that Pol II has an intrinsic capacity for translesion synthesis (TLS) that enables bypass of the CPD with or without repair. Translesion synthesis depends on the trigger loop and bridge helix, the two flexible regions of the Pol II subunit Rpb1 that participate in substrate binding, catalysis, and translocation. Substitutions in Rpb1 that promote lesion bypass in vitro increase UV resistance in vivo, and substitutions that inhibit lesion bypass decrease cell survival after UV irradiation. This work revelaed an importance of translesion transcription for cell survival upon accumulation of the unrepaired CPD lesions in genomic DNA.