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

基本信息

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
项目类别：
财政年份：
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

项目摘要

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启动的机制与哺乳动物细胞的TCR启动不同。删除Cockayne综合征B基因的删除严重抑制了哺乳动物细胞中的TCR，但删除其酵母同源物rad26仅略微损害了TCR。遗传分析强烈表明酵母中的两个替代性TCR子路口。第一个主要途径可能是由Pol II与RAD26相互作用引发的，并且取决于非必需的POL II亚基RPB4。在不存在RPB4的情况下，第二个TCR途径变得突出，并且取决于另一个非必需的POL II亚基RPB9。 RPB9介导的TCR途径的机制尚不清楚。由于缺乏RPB4/RPB9双重缺失突变体，其遗传手段的调查受到了阻碍，这可能是致命的。对酵母中RPB9依赖性途径的分析可能会提供对TCR期间与POL II相关事件的重要见解。 RPB9亚基在POL II周围的位置表明其在招募NER因素到受损部位的可能功能。 RPB9参与了多转录相关的功能，例如转录起始（开始位点的选择），转录伸长率，以及最近响应UV诱导的DNA损伤的RPB1的泛素化和降解。该亚基还与参与转录伸长和组织方法化的多种因素相互作用（例如TFIIS，TFIIE和SAGA）。这些因素中的哪个在RPB9介导的TCR途径中充当Rad26类似物。紫外线诱导的环丁烷嘧啶二聚体（CPDS）在模板DNA链损耗转录伸长延伸中，通过POL II。如果核苷酸切除修复机械未迅速删除CPD，则停滞的POL II会产生用于DNA复制的障碍，并随后进行转录。在这里，我们提供了证据表明，Pol II具有跨性别合成能力（TLS），该能力可以在有或没有修复的情况下绕过CPD。 Translesion合成取决于触发循环和桥螺旋，这是参与底物结合，催化和易位的Pol II亚基RPB1的两个柔性区域。在RPB1中的取代，可以在体内促进病变绕过体外的紫外线耐药性，并抑制病变旁路的取代可降低紫外线照射后细胞存活。这项工作启发了转化转录对于基因组DNA中未经修复的CPD病变积累而对细胞存活的重要性。