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

摘要 烷化剂替莫唑胺（TMZ）广泛用于治疗人类多形性胶质母细胞瘤 （GBM）。TMZ可诱导多种DNA损伤，包括O-6-甲基鸟嘌呤（O 6 meG）、N-7-甲基鸟嘌呤（N-7-methylguanine （7 meG）和N-3-甲基腺嘌呤（3 meA）。许多不同的DNA修复途径参与修复， 耐受烷基化损伤，包括甲基鸟嘌呤甲基转移酶（MGMT）的直接修复，碱基 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被广泛研究， NER仍然难以捉摸。我们最近使用一种新的方法研究了7 meG和3 meA损伤的全基因组修复， NMP-seq（N-甲基嘌呤测序）。我们的高分辨率修复数据显示， 转录链（TS）和非转录链（NTS）之间的不对称性在3 meA的修复中， BER缺陷的酵母细胞，其中3 meA在TS上被更快地修复。因此，我们也发现了一个 BER缺陷酵母基因组中腺嘌呤突变的强链不对称性。这些数据导致 我们假设BER缺陷细胞中持续的3 meA损伤使RNA聚合酶II停滞，并激活一个RNA聚合酶。 称为转录偶联核苷酸切除修复（TC-NER）的NER子途径，其具体 从活跃转录基因的TS去除3 meA损伤。我们建议阐明的机制， 其中TC-NER修复3 meA并防止酵母和人类细胞中基因的TS突变（Aim 1）。 我们在3 meA修复中的发现也使我们研究TC-NER是否在修复中起作用， O 6 meG的诱变。我们重新分析了TMZ治疗的MGMT缺陷GBM肿瘤中发生的突变， 其中大多数体细胞突变与TMZ诱导的O 6 meG损伤相关。引人注目的是，我们的数据显示 这些肿瘤中的TMZ特征突变在NTS上富集。这种链的不对称性 在TMZ治疗后观察到，但在初始未治疗的肿瘤中没有观察到。这些发现表明O 6 meG 在MGMT缺陷的癌细胞中，病变可能通过TC-NER途径优先从TS去除。 我们建议测试所观察到的突变链不对称性是否可以在遗传学上重现。 易处理的酵母模型系统，并表征关键TC-NER因子对O 6 meG突变的贡献。 股不对称性。此外，TC-NER在O 6 meG修复中的作用将在MGMT阴性的 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 研究已经揭示了用TMZ治疗后GBM肿瘤中显著改变的突变景观。我们 这项研究将产生第一个全基因组O 6 meG切除修复图谱，这将提供机制， GBM基因组的治疗驱动进化的证据。

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.