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: 10108465
• 负责人: Peng Mao
• 金额: $ 19.13万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 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 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10108465
• 关键词: TC; NER; REPAIR; MUTAGENESIS; DNA

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 烷化剂替莫唑胺（TMZ）广泛用于治疗人多形性胶质母细胞瘤 （GBM）。 TMZ 诱导多种 DNA 损伤，包括 O-6-甲基鸟嘌呤 (O6meG)、N-7-甲基鸟嘌呤 (7meG) 和 N-3-甲基腺嘌呤 (3meA)。许多不同的 DNA 修复途径参与修复和 耐受烷基化损伤，包括甲基鸟嘌呤甲基转移酶 (MGMT)、碱基的直接修复 切除修复（BER）、核苷酸切除修复（NER）和错配修复（MMR）。虽然功能 MGMT、BER 和 MMR 在烷基化损伤修复和耐受中的作用已得到广泛研究， NER 仍然难以捉摸。我们最近使用一种新方法检查了 7meG 和 3meA 损伤的全基因组修复 方法命名为NMP-seq（N-甲基嘌呤测序）。我们的高分辨率修复数据揭示了惊人的 3meA 修复中转录链 (TS) 和非转录链 (NTS) 之间的不对称性 BER 缺陷的酵母细胞，3meA 在 TS 上修复得更快。据此，我们还发现了一个 BER 缺陷酵母基因组中腺嘌呤突变存在强烈的链不对称性。这些数据导致 我们的假设是，BER 缺陷细胞中持续存在的 3meA 损伤会阻碍 RNA 聚合酶 II 并激活 NER 的子通路称为转录偶联核苷酸切除修复 (TC-NER)， 从活跃转录基因的 TS 中去除 3meA 损伤。我们建议通过以下方式阐明该机制： 其中 TC-NER 可修复 3meA 并防止酵母和人类细胞中基因 TS 的突变（目标 1）。 我们在 3meA 修复中的发现还促使我们研究 TC-NER 是否在修复中发挥作用，并且 O6meG 的诱变。我们重新分析了 TMZ 治疗的、MGMT 缺陷的 GBM 肿瘤中发生的突变， 大多数体细胞突变与 TMZ 诱导的 O6meG 损伤有关。引人注目的是，我们的数据表明 这些肿瘤中的 TMZ 特征突变在 NTS 上富集。该链不对称性仅 在 TMZ 治疗后观察到，但在初始、未经治疗的肿瘤中未观察到。这些发现表明 O6meG 在 MGMT 缺陷的癌细胞中，病变可能会通过 TC-NER 途径优先从 TS 中去除。 我们建议测试观察到的突变链不对称性是否可以在遗传上重现 易处理酵母模型系统，并表征关键 TC-NER 因子对 O6meG 突变的贡献 链不对称。此外，TC-NER 在 O6meG 修复中的作用将以 MGMT 阴性为特征 GBM细胞系，使用最近发布的XR-seq（切除修复测序）方法（目标2）。最近的 研究发现，TMZ 治疗后 GBM 肿瘤的突变情况发生显着改变。我们的 研究将生成第一个全基因组 O6meG 切除修复图谱，该图谱应提供机制 GBM 基因组治疗驱动进化的证据。