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

在紫外线辐射、氧化应激或DNA化学修饰引起的DNA损伤下，基因组DNA正确有效地转录成mRNA对于细胞的生存至关重要。与发生转录错误后Pol II的停滞类似，DNA损伤可以干扰复制，潜在地导致DNA突变，也会阻碍转录，影响基因组的稳定性和基因表达的调节。为了保持基因组的完整性，细胞进化出了独立的细胞策略，涉及多种DNA损伤修复和DNA损伤耐受机制。非大块DNA损伤优先通过碱基切除修复(BER)途径修复，而导致DNA大变形的损伤，如紫外光诱导的环丁烷嘧啶二聚体(CPDS)/顺铂加合物和氧化环嘌呤，则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，但一些病变逃脱了检测，为细胞持续的DNA和RNA合成提出了挑战。在复制过程中，跨病变DNA合成[TLS]可以减轻DNA损伤的有害影响。在TLS过程中，高保真的复制型DNA聚合酶瞬间切换到专门的跨病变DNA聚合酶，这种DNA聚合酶可以在更宽敞的活性部位内容纳巨大的病变，从而使它们能够旁路。最近，我们证明了酵母POL II采用了一种独特的CPD旁路机制(称为A规则)，涉及其活性中心(Rpb1亚基中的可移动触发环结构域)的构象灵活性，有助于调节巨大的病变。我们最近将这一项目扩展到Pol II旁路块状环嘌呤(CyPn)转录基因的氧化损伤，并通过哺乳动物(小牛胸腺，CT)Pol II的巨大损伤来分析RNA聚合酶跨病变合成(RTLS)。我们还计划在体外识别和测试参与RTLS和酵母和哺乳动物细胞TCR启动的蛋白质因子。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.