N6-methyladenosine (m6A) Interplays with RNA and DNA Damage to Regulate DNA Repair

N6-甲基腺苷（m6 A）是mRNA中最常见的修饰，并调节基因表达。 m6 A谱的调节与癌症进展和化疗药物抗性相关。 然而，目前尚不清楚m6 A如何参与这些过程。了解潜在机制 是发现用于癌症治疗和诊断的新药物靶点和生物标志物的关键。最近的研究 已经指出m6 A在调节RNA引导的DNA修复中的新作用，表明它可以介导癌症 通过与DNA损伤和修复的相互作用来发展。我们最近发现一个m6 A位于 DNA聚合酶β（Pol β）转录产物上的终止密码子被氧化性RNA碱基损伤所消除 由于环境毒物溴酸钾（KBrO 3）的诱导，使其沉积转移到河流的上游， Pol β转录本。我们假设m6 A与RNA和DNA碱基损伤相互作用来调节DNA修复。 为了验证这个假设，我们将追求两个具体目标。目的1是确定m6 A谱和丰度是否 可以通过癌细胞中RNA和DNA碱基损伤来调节。首先，我们将确定氧化RNA和 DNA碱基损伤可破坏癌细胞中Pol β转录物上的m6 A谱。这将在 用KBrO 3处理的HeLa细胞。我们将确定m6 A谱是否可以通过氧化RNA碱基 使用DNA-RNA免疫沉淀法在Pol β转录物上的损伤、8-oxoG和脱碱基位点（AP位点） （DRIP）介导的链断裂介导的RNA修饰谱分析（DRIP-SBRM）。然后我们将决定 如果DNA碱基损伤、8-oxodGs和Pol β DNA模板上的AP位点可以改变m6 A谱， 使用DRIP介导的链断裂介导的DNA修饰分析（DRIP- SBDM）。其次，我们将确定RNA和DNA碱基损伤是否会改变活性和底物结合 m6 A写入器和擦除器、<$L3/<$L14和FTO的亲和力。目的2是确定m6 A是否可以改变 RNA碱基损伤的积累来调节癌细胞中的DNA修复。首先我们要确定 在胃L3下，m6 A可以修饰Pol β转录物上的氧化DNA和RNA碱基损伤 缺乏HeLa细胞。m6 A对DNA和RNA碱基损伤的影响 将使用具有或不具有胃L3基因敲除的HeLa细胞测定KBrO 3。m6 A和 对Pol β转录本及其模板DNA的氧化碱基损伤将是相关的。第二，我们将确定 如果关键BER酶的活性和底物结合亲和力可以在m6 A和RNA中调节， 损伤位置依赖方式。这项研究将证明新的概念，epitranscriptomic修改 与RNA和DNA碱基损伤相互作用以调节癌细胞中的DNA修复。结果将揭示一个新的 m6 A与氧化性RNA和DNA损伤相互作用以调节癌症基础DNA修复的范例 进展因此，这项研究将有助于发现基于RNA的治疗，诊断和治疗的新靶点。 预防环境诱发的癌症。

N6-methyladenosine (m6A) is the most common modification in mRNA and regulates gene expression. Modulation of m6A profiles is associated with cancer progression and chemotherapeutic drug resistance. However, it remains unknown how m6A is involved in the processes. Understanding the underlying mechanisms is the key to the discovery of new drug targets and biomarkers for cancer therapy and diagnosis. Recent studies have pointed to a new role of m6A in regulating RNA-guided DNA repair suggesting that it can mediate cancer development by interplaying with DNA damage and repair. We recently found that an m6A located next to the stop codon on the transcript of DNA polymerase β (Pol β) was eliminated by oxidative RNA base damage induced by the environmental toxicant, potassium bromate (KBrO3), shifting its deposition to the upstream of the Pol β transcript. We hypothesize that m6A interplays with RNA and DNA base damage to regulate DNA repair. To test this hypothesis, we will pursue two Specific Aims. Aim 1 is to determine if m6A profiles and abundance can be modulated by RNA and DNA base damage in cancer cells. First, we will determine if oxidative RNA and DNA base damage can disrupt the profiles of m6A on the Pol β transcript in cancer cells. This will be tested in HeLa cells treated with KBrO3. We will determine if m6A profiles can be modulated by oxidative RNA base damage, 8-oxoG, and abasic sites (AP sites) on the Pol β transcript using DNA-RNA immunoprecipitation (DRIP)-mediated strand break-mediated RNA modification profiling assay (DRIP-SBRM). We will then determine if the m6A profiles can be altered by DNA base damage, 8-oxodGs, and AP sites on the DNA template of Pol β transcript using the DRIP-mediated strand break-mediated DNA modification profiling assay (DRIP- SBDM). Second, we will determine if RNA and DNA base damage can alter the activity and substrate binding affinity of the m6A writer and eraser, METTL3/METTL14 and FTO. Aim 2 is to determine if m6A can alter the accumulation of RNA base damage to modulate DNA repair in cancer cells. First, we will determine if the profiles of oxidative DNA and RNA base damage on the Pol β transcript can be modified by m6A under METTL3 deficiency in HeLa cells. The effects of m6A on the profiles of oxidative DNA and RNA base damage induced by KBrO3 will be determined using HeLa cells with or without METTL3 gene knockdown. The profiles of m6A and oxidative base damage on the Pol β transcript and its template DNA will be correlated. Second, we will determine if the activity and substrate binding affinity of the key BER enzymes can be modulated in an m6A and RNA damage location-dependent manner. The study will prove the new concept that epitranscriptomic modifications interplay with RNA and DNA base damage to regulate DNA repair in cancer cells. The results will reveal a novel paradigm for m6A to interplay with oxidative RNA and DNA damage to modulate DNA repair that underlies cancer progression. Thus, the study will facilitate the discovery of new targets for RNA-based treatment, diagnosis, and prevention of environmentally-induced cancer.