Mechanism of Transcription-coupled DNA Repair and its Impact on Cancer Mutations

转录偶联DNA修复机制及其对癌症突变的影响

• 批准号: 10660150
• 负责人: Peng Mao
• 金额: $ 32.16万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-04 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10660150
• 关键词: ATP phosphohydrolase; Address; Alkylation; Apoptosis; Base Excision Repairs; Binding; Cells; Chromatin; Cockayne Syndrome; Code; Complex; Coupled; Coupling; DNA; DNA Damage; DNA Repair; DNA lesion; DNA mapping; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Elongation Factor; Esophageal Adenocarcinoma; Esophageal Neoplasms; Eukaryota; Excision; Foundations; Genes; Genetic Transcription; Genome; Human; Human Genome; Investigation; Knowledge; Lesion; Malignant Neoplasms; Maps; Mediating; Methods; Molecular Conformation; Mutagenesis; Mutation; Mutation Analysis; Nucleosomes; Nucleotide Excision Repair; Nucleotides; Orthologous Gene; Outcome; Pathway interactions; Peptide Initiation Factors; Play; Polymerase; Proteins; Pyrimidine Dimers; RNA Polymerase II; Reaction; Repair Complex; Research; Resolution; Role; SWI2/SNF2; Somatic Mutation; Testing; Transcription Initiation; Transcription-Coupled Repair; Transcriptional Elongation Factors; UV induced; Yeasts; base; cancer genome; chromatin remodeling; genome-wide; improved; innovation; insight; methylpurine; mutant; nervous system disorder; oxidative DNA damage; oxidative damage; prevent; recruit; repair enzyme; repaired; transcription factor TFIIH; ultraviolet damage; yeast genome

ABSTRACT Elongating RNA polymerase II (Pol II) can be blocked by a variety of DNA damage. The stalled Pol II prevents passage of other RNA and DNA polymerases and blocks exposure of damage to repair proteins, leading to apoptosis or mutagenesis. To avoid these detrimental outcomes, cells activate several mechanisms, including transcription-coupled nucleotide excision repair (TC-NER), to rescue the stalled Pol II. In human cells, the Cockayne Syndrome B (CSB) protein is believed to bind to damage-stalled Pol II and initiate TC-NER. However, there is a critical gap in knowledge concerning how CSB switches Pol II from elongation to a form amenable to DNA repair. Additionally, TC-NER is best known to repair helix-distorting (bulky) DNA lesions, but whether it also repairs non-bulky base damage that occurs more frequently in living cells is poorly understood. To address these important questions, we developed genome-wide and single-nucleotide resolution sequencing methods to map DNA lesions, including bulky cyclobutane pyrimidine dimers (CPDs; UV damage) and non-bulky N-methylpurines (NMPs; alkylation damage). Notably, our CPD-seq data indicates that yeast Rad26, an ortholog of CSB, functions in displacing the transcription elongation factor, Spt4-Spt5, from the stalled Pol II. This function of Rad26 is mainly required for gene coding regions downstream of the first (+1) nucleosome. The eviction of Spt4-Spt5 likely disrupts the closed conformation of the Pol II complex, thereby switching Pol II from elongation to repair. Furthermore, we identified a subset of genes in which TC-NER across the entire coding region was independent of Rad26, suggesting both Rad26-dependent and independent TC-NER mechanisms function in the yeast genome. How CSB functions and whether CSB- independent genes exist in human cells are unclear. In this proposal, we will utilize an improved CPD-seq method to generate genome-wide TC-NER profiles in CSB-proficient and deficient human cells. Comparison of the two repair maps will help identify CSB-dependent and independent genes and guide investigation into their underlying mechanisms (Aim 1). The binding of CSB to Pol II is the first step for TC-NER. Damage removal requires the assembly of a large nucleotide excision repair (NER) complex on DNA. TFIIH is the next crucial NER factor following CSB. Aim 2 will elucidate TFIIH recruitment to test the hypothesis that CSB-mediated Spt4-Spt5 displacement leads to TFIIH binding to the stalled Pol II. Moreover, our NMP-seq data indicates that TC-NER also repairs non-bulky alkylation lesions, suggesting TC-NER targets a broader spectrum of DNA damage than currently appreciated. Aim 3 is based on this intriguing finding and will focus on TC-NER of oxidative base damage, which is the most frequent endogenous damage with a profound role in cancer mutagenesis. Hence, these proposed studies will use innovative approaches and significantly improve TC- NER research by offering genome-wide insights for both bulky and non-bulky lesions.

摘要 延伸RNA聚合酶II（Pol II）可被多种DNA损伤阻断。停滞的Pol II阻止了 通过其他RNA和DNA聚合酶，并阻止损伤暴露于修复蛋白，导致 细胞凋亡或诱变。为了避免这些有害的结果，细胞激活几种机制，包括 转录偶联核苷酸切除修复（TC-NER），以挽救停滞的Pol II。在人类细胞中， Cockayne综合征B（CS B）蛋白被认为与损伤停滞的Pol II结合并启动TC-NER。 然而，关于CSB如何将Pol II从伸长转换为一种形式， 可以进行DNA修复此外，TC-NER最为人所知的是修复螺旋扭曲（庞大）DNA损伤，但 它是否也修复在活细胞中更频繁发生的非大体积碱基损伤，目前还知之甚少。 为了解决这些重要问题，我们开发了全基因组和单核苷酸分辨率 测序方法来绘制DNA损伤，包括庞大的环丁烷嘧啶二聚体（CPD; UV损伤） 和非大体积N-甲基嘌呤（NMP;烷基化损伤）。值得注意的是，我们的CPD-seq数据表明， Rad 26是CSB的直系同源物，其功能是将转录延伸因子Spt 4-Spt 5从转录延伸因子中置换出来。 停滞的波尔二号Rad 26的这种功能主要是第一个（+1） 核小体Spt 4-Spt 5的驱逐可能破坏Pol II复合物的闭合构象，从而 将Pol II从伸长切换到修复。此外，我们确定了一个基因子集，其中TC-NER 在整个编码区中的表达不依赖于Rad 26，这表明Rad 26依赖性和 独立的TC-NER机制在酵母基因组中起作用。CSB如何运作，CSB是否- 人类细胞中是否存在独立的基因尚不清楚。在本提案中，我们将利用改进的CPD序列 在CSB-熟练的和缺乏的人类细胞中产生全基因组TC-NER谱的方法。比较 这两个修复图谱将有助于识别CSB依赖性和独立性基因，并指导对其的研究 基本机制（目标1）。CSB与Pol II的结合是TC-NER的第一步。损伤去除 需要在DNA上组装大的核苷酸切除修复（NER）复合物。TFIIH是下一个关键 CSB之后的净入学率因素。目的2将阐明TFIIH募集，以检验CSB介导的TFIIH募集的假设。 Spt 4-Spt 5置换导致TFIIH与停滞的Pol II结合。此外，我们的NMP-seq数据表明， TC-NER还修复非大体积烷基化病变，表明TC-NER靶向更广泛的DNA 比目前估计的损失更大。目标3是基于这一有趣的发现，并将重点放在 氧化性碱基损伤，这是最常见的内源性损伤，在癌症中具有深远的作用 诱变因此，这些拟议的研究将采用创新的方法，大大提高TC- NER研究通过提供大体积和非大体积病变的全基因组见解。