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.

酿酒葡萄球菌的TCR启动机制不同于哺乳动物细胞的TCR启动机制。Cockayne综合征B组基因的缺失严重抑制哺乳动物细胞的TCR，而其酵母同源物Rad26的缺失仅轻微损害TCR。遗传分析强烈表明酵母中有两种不同的TCR亚通路。第一种主要途径可能是由Pol II与Rad26相互作用引发的，并且依赖于非必需的Pol II亚基Rpb4。在缺乏Rpb4的情况下，第二个TCR通路变得突出，并依赖于另一个非必需的Pol II亚基Rpb9。rpb9介导的TCR通路的机制尚不清楚。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阻碍。酵母中rpb9依赖通路的分析可能为TCR过程中Pol ii相关事件的研究提供重要的见解。Rpb9亚基在Pol II周围的位置表明它可能在将NER因子招募到受损部位中起作用。Rpb9参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，最近还参与了rpb1的泛素化和降解，以响应紫外线诱导的DNA损伤。该亚基还与转录延伸和组织修饰的过多因子相互作用（如TFIIS， TFIIE和SAGA）。这些因子中哪一个在rpb9介导的TCR通路中作为Rad26类似物仍有待确定。今年（2012年），该项目在《分子细胞》上发表了一篇论文，展示了酵母Pol II通过CPD病变进行转录的机制。紫外光诱导的环丁烷嘧啶二聚体（CPDs）在模板DNA链上被Pol II阻碍转录延伸。如果核苷酸切除修复机制不能及时移除cpd，停滞的Pol II就会为DNA复制和随后的转录制造障碍。在这里，我们提出的证据表明，Pol II具有翻译合成（TLS）的内在能力，可以在有或没有修复的情况下绕过CPD。翻译合成依赖于触发环和桥螺旋，这是Pol II亚基Rpb1的两个柔性区域，参与底物结合、催化和易位。体外促进病变旁路的Rpb1取代增加了体内对紫外线的抵抗力，抑制病变旁路的取代降低了紫外线照射后细胞的存活率。这项工作揭示了翻译转录在基因组DNA中未修复的CPD病变积累时对细胞存活的重要性。