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

基因组DNA到mRNA的忠实和有效的转录是至关重要的细胞生存的DNA损伤所造成的紫外线照射，氧化应激或化学DNA修饰。类似于Pol II在转录错误后的停滞，DNA损伤可以干扰复制，可能导致DNA突变，并且还阻碍转录，影响基因组稳定性和基因表达的调控。为了保持基因组的完整性，细胞已经进化出涉及多种DNA损伤修复和DNA损伤耐受机制的单独的细胞策略。非大体积DNA损伤优先通过碱基切除修复（BER）途径修复，而引起大的DNA畸变的损伤，如UV光诱导的环丁烷嘧啶二聚体（CPD）/顺铂加合物和氧化性环嘌呤主要受到核苷酸切除修复（NER）途径的影响。尽管正在进行修复，但一些病变仍无法被检测到，这给细胞继续合成DNA和RNA带来了挑战。在复制过程中，DNA损伤的有害影响可以通过跨损伤DNA合成[TLS]来减轻。在TLS过程中，高保真复制DNA聚合酶被瞬时转换为专门的跨病变DNA聚合酶，该跨病变DNA聚合酶可以在更宽敞的活性位点内容纳庞大的病变，从而使它们能够绕过。最近，我们证明，酵母Pol II采用一种独特的机制CPD旁路（称为A规则），涉及其活性中心（Rpb 1亚基中的移动的触发环结构域）的构象灵活性，促进大体积病变的适应。我们最近扩大了这个项目，以Pol II旁路的庞大的环嘌呤（CyPn）的氧化损伤的转录基因和RTLS（RNA聚合酶translesion合成）的分析，通过庞大的病变由哺乳动物（小牛胸腺，CT）Pol II。我们还计划在体外鉴定和测试参与RTLS和酵母和哺乳动物细胞中TCR起始的蛋白质因子。我们已经发现哺乳动物TFIIF在体外通过CT Pol II显著促进RTLS。

The faithful and efficient transcription of genomic DNA into mRNA is crucial for cell survival under DNA damage caused by UV irradiation, oxidative stress or chemical DNA modifications. Similar to arrest of Pol II after making transcription error, DNA lesions can interfere with replication, potentially causing mutations in DNA, and also hinder transcription, affecting genome stability and regulation of gene expression. To maintain genomic integrity, cells have evolved separate cellular strategies involving multiple DNA damage repair and DNA damage tolerance mechanisms. Non-bulky DNA lesions are preferentially repaired by the base excision repair (BER) pathway while damages that cause large DNA distortion, such as UV light-induced cyclobutane pyrimidine dimers (CPDs)/cisplatin adducts and oxidative cyclopurines are primarily subject to the nucleotide excision repair (NER) pathway. Despite ongoing repair, some lesions escape detection, presenting the cell with a challenge for continued DNA and RNA synthesis. During replication, the deleterious effect of DNA lesions can be alleviated by translesion DNA synthesis [TLS]. During TLS, the high-fidelity replicative DNA polymerases are switched transiently to specialized translesion DNA polymerases that can accommodate bulky lesions within a more spacious active site, thus enabling their bypass. Recently, we demonstrated that yeast Pol II employs a distinct mechanism for CPD bypass (called A-rule) involving a conformational flexibility of its active center (the mobile trigger loop domain in Rpb1 subunit) facilitating accommodation of bulky lesions. We recently expanded this project to Pol II bypass of bulky cyclopurine (CyPn) oxidative damages in transcribed genes and to analysis of RTLS (RNA polymerase translesion synthesis) through the bulky lesions by mammalian (calf thymus, CT) Pol II. We also plan to identify and test in vitro protein factors involved in RTLS and in initiation of TCR in yeast and mammalian cells. We have already found that mammalian TFIIF significantly promotes RTLS by CT Pol II in vitro.