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

转录偶联 DNA 修复 (TCR) 初始步骤的机制

• 批准号: 8763394
• 负责人: MIKHAIL KASHLEV
• 金额: $ 54.38万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763394
• 关键词: Active Sites; Address; Aging; Base Excision Repairs; Binding; Biological Models; Bypass; Cell Survival; Cells; Chemicals; Chromatin; Cisplatin; Complex; Coupled; DNA; DNA Damage; DNA Modification Process; DNA Polymerase II; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA lesion; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; DNA-dependent ATPase; Detection; Development; Enzymes; Escherichia coli; Eukaryota; Excision; Family; Genes; Genetic; Genetic Transcription; Genome; Genomics; Goals; Growth; In Vitro; Lead; Lesion; Mammalian Cell; Mediation; Messenger RNA; Molecular; Nature; Nucleosomes; Nucleotide Excision Repair; Oxidative Stress; Pathway interactions; Play; Polymerase; Positioning Attribute; Process; Proteins; Protocols documentation; Pyrimidine Dimers; RNA chemical synthesis; Recombinant DNA; Recruitment Activity; Research; Resistance; Role; Saccharomyces cerevisiae; Signal Transduction; Site; Stress; System; Thymidine; Transcription Elongation; Transcription Initiation; Transcription-Coupled Repair; Type II Cockayne Syndrome; UV induced; Ultraviolet Rays; Variant; Work; Yeasts; adduct; base; chemotherapeutic agent; crosslink; dimer; helicase; member; novel; originality; repaired; success; transcription factor TFIIH; tumor; tumorigenesis; ubiquitin ligase; ultraviolet irradiation

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. 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. In the past 4 years our work on transcription-coupled DNA repair (TCR) revealed that yeast Pol II has an intrinsic capability to bypass UV-induced CPDs as an alternative pathway to its preferential repair by TC-NER. We defined this process as RNA polymerase translesion synthesis (RTLS, here and herein) to distinguish it from the regular TLS by DNA polymerases. We further demonstrated that the efficiency of the CPD bypass by Pol II correlates with increased UV cell resistance. Most importantly, our most recent unpublished results revealed that mammalian Pol II employs the similar mechanism to negotiate with the CPDs in vitro. To address the functional correlation between RTLS and TC-NER, we will investigate the mechanism for strand specific repair in various genetic backgrounds in yeast as a model system. We will expand Project 2 to transcription across the other types of DNA lesions including cyclopurine lesions (cyclo-dA and cyclo-dG). Our preliminary results showed that yeast and mammalian Pol II are capable of transcription through cyclo-dA in vitro by using a mechanism, which is strikingly similar to transcription across the CPDs. We will continue elucidation of the role of Rpb4/7 and Rpb9 subunits of Pol II in initiation of TCR and will search for repair proteins specific to Rpb4/7- and Rpb9-dependent TCR pathways. In the long term, this project will be merged with "Transcription Fidelity" project to evaluate an impact of faithful and error-prone transcription to genome integrity during normal growth and endogenous/exogenous stresses caused by DNA damage, tumorigenesis and aging.

基因组 DNA 忠实、高效地转录为 mRNA，对于细胞在紫外线照射、氧化应激或化学 DNA 修饰引起的 DNA 损伤下的生存至关重要。为了维持基因组完整性，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。非大体积 DNA 损伤优先通过碱基切除修复 (BER) 途径进行修复，而导致大量 DNA 变形的损伤，例如紫外线诱导的环丁烷嘧啶二聚体 (CPD)/顺铂加合物和氧化环嘌呤则主要受到核苷酸切除的影响修复（NER）途径。尽管修复不断进行，但一些病变仍无法被发现，这给细胞持续合成 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 聚合酶会瞬时切换为专门的跨损伤 DNA 聚合酶，可以在更宽敞的活性位点内容纳大块损伤，从而实现绕过。在过去 4 年中，我们在转录偶联 DNA 修复 (TCR) 方面的工作表明，酵母 Pol II 具有绕过紫外线诱导的 CPD 的内在能力，作为 TC-NER 优先修复的替代途径。我们将此过程定义为 RNA 聚合酶跨损伤合成（RTLS，此处和此处），以将其与 DNA 聚合酶的常规 TLS 区分开来。我们进一步证明，Pol II 的 CPD 旁路效率与 UV 电池电阻的增加相关。最重要的是，我们最近未发表的结果表明，哺乳动物 Pol II 采用类似的机制在体外与 CPD 进行协商。为了解决 RTLS 和 TC-NER 之间的功能相关性，我们将以酵母作为模型系统研究各种遗传背景下链特异性修复的机制。我们将项目 2 扩展到其他类型 DNA 损伤的转录，包括环嘌呤损伤（cyclo-dA 和 cyclo-dG）。我们的初步结果表明，酵母和哺乳动物 Pol II 能够在体外通过 cyclo-dA 进行转录，其机制与跨 CPD 的转录惊人相似。我们将继续阐明 Pol II 的 Rpb4/7 和 Rpb9 亚基在 TCR 启动中的作用，并将寻找 Rpb4/7 和 Rpb9 依赖性 TCR 途径特异的修复蛋白。从长远来看，该项目将与“转录保真度”项目合并，以评估在正常生长以及DNA损伤、肿瘤发生和衰老引起的内源/外源应激期间，忠实且易错的转录对基因组完整性的影响。