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: 8553035
• 负责人: MIKHAIL KASHLEV
• 金额: $ 61.52万
• 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/8553035
• 关键词: Active Sites; Address; Binding; Bypass; Catalysis; Cell Survival; Cells; Chromatin; Cisplatin; Complex; Coupled; DNA; DNA Damage; DNA Polymerase II; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA lesion; DNA-dependent ATPase; Development; Enzymes; Escherichia coli; Eukaryota; Event; Excision; Family; Genes; Genetic; Genetic Transcription; Genomics; Goals; Homologous Gene; In Vitro; Investigation; Lead; Lesion; Location; Mammalian Cell; Manuscripts; Mediating; Molecular; Nature; Nucleosomes; Nucleotide Excision Repair; Pathway interactions; Play; Polymerase; Positioning Attribute; Proteins; Protocols documentation; Publications; Pyrimidine Dimers; Recombinant DNA; Recruitment Activity; Research; Resistance; Role; SAGA; Saccharomyces cerevisiae; Signal Transduction; Site; System; Thymidine; Transcription Elongation; Transcription Initiation; Transcription-Coupled Repair; Type II Cockayne Syndrome; UV induced; UV induced DNA damage; Ubiquitination; Variant; Work; Yeasts; adduct; analog; base; chemotherapeutic agent; crosslink; dimer; flexibility; genetic analysis; helicase; in vivo; insight; member; mutant; novel; originality; repaired; response; success; transcription factor S-II; transcription factor TFIIE; transcription factor TFIIH; tumor; ubiquitin ligase; ultraviolet irradiation

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.

酿酒酵母中TCR起始的机制不同于哺乳动物细胞中TCR起始的机制。虽然科凯恩综合征 B 组基因的缺失会严重抑制哺乳动物细胞中的 TCR，但其酵母同源物 Rad26 的缺失仅轻微损害 TCR。遗传分析强烈表明酵母中有两种替代的 TCR 子通路。第一条占主导地位的途径可能是由 Pol II 与 Rad26 相互作用启动的，并且依赖于非必需的 Pol II 亚基 Rpb4。第二条 TCR 途径在 Rpb4 缺失的情况下变得突出，并且依赖于另一个非必需的 Pol II 亚基 Rpb9。 Rpb9 介导的 TCR 途径的机制尚不清楚。由于缺乏可能致命的 RPB4/RPB9 双缺失突变体，通过遗传学手段对其进行研究受到了阻碍。对酵母中 Rpb9 依赖性途径的分析可能为 TCR 期间 Pol II 相关事件提供重要的见解。 Rpb9 亚基位于 Pol II 周边的位置表明其可能具有将 NER 因子招募到受损位点的功能。 Rpb9 参与多转录相关功能，例如转录起始（起始位点的选择）、转录延伸，以及最近响应紫外线诱导的 DNA 损伤而导致 rpb1 泛素化和降解。该亚基还与多种参与转录延伸和组蛋白修饰的因子（如 TFIIS、TFIIE 和 SAGA）相互作用。这些因子中的哪一个在 Rpb9 介导的 TCR 途径中充当 Rad26 类似物仍有待确定。今年（2012 年）该项目的手稿发表在《分子细胞》杂志上，展示了酵母 Pol II 通过CPD 病变。模板 DNA 链中紫外线诱导的环丁烷嘧啶二聚体 (CPD) 可阻止 Pol II 的转录延伸。如果核苷酸切除修复机制不能及时去除 CPD，停滞的 Pol II 就会为 DNA 复制和后续几轮转录造成障碍。在这里，我们提供的证据表明，Pol II 具有跨损伤合成 (TLS) 的内在能力，可以在有或没有修复的情况下绕过 CPD。跨损伤合成取决于触发环和桥螺旋，这两个 Pol II 亚基 Rpb1 的灵活区域参与底物结合、催化和易位。在体外促进病变旁路的 Rpb1 取代会增加体内的紫外线抵抗力，而抑制病变旁路的取代则会降低紫外线照射后的细胞存活率。这项工作揭示了基因组 DNA 中未修复的 CPD 损伤积累后，跨损伤转录对于细胞生存的重要性。