TC-NER IN THE REPAIR AND MUTAGENESIS OF DNA ALKYLATION DAMAGE

TC-NER 在 DNA 烷基化损伤的修复和诱变中的作用

• 批准号: 9508890
• 负责人: Peng Mao
• 金额: $ 22.95万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9508890
• 关键词: 3-methyladenine; Adenine; Aftercare; Alkylating Agents; Alkylation; Base Excision Repairs; Cell Line; Cells; DNA; DNA Alkylation; DNA Ligation; DNA Repair; DNA Repair Pathway; DNA glycosylase; DNA lesion; DNA sequencing; Data; Dinucleoside Phosphates; Evolution; Excision Repair; Exposure to; Foundations; Genes; Genetic Transcription; Genome; Glioblastoma; Glioma; Homologous Gene; Human; Human Genome; Lead; Lesion; MGMT gene; Malignant Neoplasms; Maps; Measures; Methods; Methyl Methanesulfonate; Methyltransferase; Mismatch Repair; Modeling; Mutagenesis; Mutation; Names; Nucleotide Excision Repair; O-(6)-methylguanine; Pathway interactions; Patients; Pattern; Play; Publishing; RNA Polymerase II; Recurrence; Resistance; Resolution; Role; Shapes; Somatic Mutation; Testing; Therapeutic; Transcription-Coupled Repair; UV induced; Yeast Model System; Yeasts; base; cancer cell; cancer genome; cell type; chemotherapeutic agent; chemotherapy; clinically relevant; cytotoxic; density; endonuclease; genome-wide; genome-wide analysis; insight; melanoma; methylpurine; mutant; next generation; novel; prevent; repaired; temozolomide; tumor

ABSTRACT The alkylating agent temozolomide (TMZ) is widely used for the treatment of human glioblastoma multiforme (GBM). TMZ induces a variety of DNA lesions, including O-6-methylguanine (O6meG), N-7-methylguanine (7meG), and N-3-methyladenine (3meA). Many different DNA repair pathways are involved in the repair and tolerance of alkylation damage, including direct repair by methyl-guanine methyltransferase (MGMT), base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). Although the functions of MGMT, BER, and MMR in alkylation damage repair and tolerance have been extensively studied, the role of NER remains elusive. We recently examined genome-wide repair of 7meG and 3meA lesions using a novel method named as NMP-seq (N-methylpurine sequencing). Our high-resolution repair data revealed a striking asymmetry between the transcribed strand (TS) and the non-transcribed strand (NTS) in the repair of 3meA in BER-deficient yeast cells, with 3meA being repaired more rapidly on the TS. Accordingly, we also found a strong strand asymmetry in adenine mutations across the genome of BER-deficient yeast. These data lead to our hypothesis that the persisting 3meA lesions in BER-deficient cells stall RNA Polymerase II and activate a subpathway of NER known as transcription-coupled nucleotide excision repair (TC-NER), which specifically removes 3meA lesions from the TS of actively transcribed genes. We propose to elucidate the mechanism by which TC-NER repairs 3meA and prevents mutations on the TS of genes in yeast and human cells (Aim 1). Our findings in 3meA repair also led us to investigate whether TC-NER plays a role in the repair and mutagenesis of O6meG. We reanalyzed mutations occurring in TMZ-treated, MGMT-deficient GBM tumors, in which most somatic mutations are associated with TMZ-induced O6meG lesions. Strikingly, our data indicate that the TMZ signature mutations in these tumors are enriched on the NTS. This strand asymmetry was only observed following TMZ treatment but not in initial, untreated tumors. These findings suggest that O6meG lesions are preferentially removed from the TS in MGMT-deficient cancer cells, likely by the TC-NER pathway. We propose to test whether the observed mutational strand asymmetry can be recapitulated in the genetically tractable yeast model system, and characterize the contributions of key TC-NER factors to O6meG mutational strand asymmetry. Additionally, the role of TC-NER in O6meG repair will be characterized in a MGMT-negative GBM cell line, using the recently published XR-seq (Excision Repair Sequencing) method (Aim 2). Recent studies have uncovered significantly altered mutation landscape in GBM tumors post-treatment with TMZ. Our study will generate the first genome-wide O6meG excision repair map, which should provide mechanistic evidence for therapy-driven evolution of the GBM genome.

抽象的 烷基化剂替莫唑胺（TMZ）广泛用于治疗人胶质母细胞瘤多形 （GBM）。 TMZ诱导多种DNA病变，包括O-6-甲基鸟嘌呤（O6meg），N-7-甲基鸟嘌呤 （7meg）和N-3-甲基趋化（3mea）。维修中涉及许多不同的DNA修复途径 烷基化损伤的耐受性，包括通过甲基 - 瓜氨酸甲基转移酶（MGMT）直接修复的碱基 切除修复（BER），核苷酸切除修复（NER）和不匹配修复（MMR）。虽然功能 MGMT，BER和MMR在烷基化损伤修复和耐受性方面进行了广泛研究， ner仍然难以捉摸。我们最近使用一种新型检查了7meg和3mea病变的全基因组修复 方法称为NMP-SEQ（N-甲基嘌呤测序）。我们的高分辨率维修数据显示出惊人的 在修复3-MEA中，转录链（TS）和非转录链（NTS）之间的不对称性 缺乏Ber的酵母细胞，TS上的3mea修复更快。因此，我们还发现了 在缺乏Ber酵母基因组的腺嘌呤突变中，强链不对称性。这些数据导致 我们的假设是，在缺乏BER的细胞中持续存在的3MEA病变失速RNA聚合酶II并激活A NER被称为转录耦合的核苷酸切除修复（TC-NER）的子路口，特别是 从主动转录基因的TS中去除3MEA病变。我们建议通过 TC-NER可以修复3MEA，并防止酵母和人类细胞中基因TS的突变（AIM 1）。 我们在3MEA维修中的发现也使我们调查了TC-NER在维修中是否起作用，并且 O6meg的诱变。我们重新分析了在TMZ处理的MGMT缺乏GBM肿瘤中发生的突变 大多数体细胞突变与TMZ诱导的O6meg病变有关。令人惊讶的是，我们的数据表明 这些肿瘤中的TMZ特征突变富集在NTS上。这种不对称性仅是 在TMZ处理后观察到，但未在初始未处理的肿瘤中观察到。这些发现表明O6meg 可能通过TC-NER途径从MGMT缺陷型癌细胞中的TS中除去病变。 我们建议测试在遗传上是否可以概括观察到的突变不对称性 可处理的酵母模型系统，并表征关键TC-NER因子对O6meg突变的贡献 链不对称。另外，将在MGMT阴性中表征TC-NER在O6meg修复中的作用 GBM细胞系，使用最近发表的XR-Seq（切除修复测序）方法（AIM 2）。最近的 研究后，研究发现了与TMZ治疗后GBM肿瘤中突变景观的显着改变。我们的 研究将生成第一个全基因组O6meg切除修复图，该图应提供机械 GBM基因组治疗驱动的演变的证据。