Molecular Mechanism of Mammalian DNA Excision Repair and the Circadian Clock

哺乳动物DNA切除修复和生物钟的分子机制

• 批准号: 10687262
• 负责人: AZIZ SANCAR
• 金额: $ 100.27万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10687262
• 关键词: 3-Dimensional; Area; Biochemical; Carcinogens; Chromatin; Chronotherapy; Cisplatin; Colorectal Cancer; DNA; DNA Damage; DNA Repair; DNA mapping; Diptera; Drosophila genus; Excision Repair; Feedback; Future; Genes; Genetic Transcription; Genome; Genomics; Goals; Health; Human; In Vitro; Insecta; Kidney; Link; Liver; Lung; Maps; Methods; Modeling; Molecular; Mus; Nucleotide Excision Repair; Nucleotides; Pharmaceutical Preparations; Process; Regimen; Regulation; Research; Resolution; Spottings; System; Technology; Therapeutic; Time; Tissues; Transcription-Coupled Repair; Translations; Work; cancer prevention; cancer therapy; cancer type; chemotherapy; circadian pacemaker; cryptochrome; epigenomics; experimental study; genetic approach; genome-wide; innovation; novel; oxaliplatin; protein purification; reconstitution; repaired

PROJECT SUMMARY/ABSTRACT We work on molecular mechanisms of nucleotide excision repair and the mammalian circadian clock. We have recently made both the technological and mechanistic progress in both of these areas and obtained direct evidence of inter-connectedness of these two fields. Our findings in both fields are directly applicable to human health. We will apply these new advances for the following objectives: (1) We have developed higher resolution versions of our original XR-seq method for genome-wide single- nucleotide resolution mapping of repair of all DNA damage that is processed by nucleotide excision repair, including damage induced by carcinogens and chemotherapeutic drugs. In addition, we developed Damage-seq methods for similarly genome-wide single nucleotide resolution mapping of DNA damage. We have used the combination of the two methods to discover repair hotspots and coldspots that do not overlap damage hotspots or coldspots and have gained novel information on genome 3D and repair. We will continue characterizing these features to link repair to epigenomic markers, 3D genome organization, chromatin states, and replication timing. XR-seq has also enabled us to discover a novel transcription-coupled repair mechanism in Drosophila and other insects in the order Diptera. We will use biochemical and genetic approaches to solve the mechanism of this novel repair system. (2) We will define the molecular mechanism of the mammalian circadian clock. Recently, we demonstrated that Cryptochrome (CRY), and not Period (PER), is the repressor in the mammalian transcription-translation feedback loop (TTFL), and that PER acts either as a repressor or an activator, depending on the particular gene, in a CRY-dependent manner. We will carry out experiments to reconstitute this model in an in vitro system with purified proteins. (3) Circadian clock, cancer, and chemotherapy. For the first time, we have been able to map both damage formation by cisplatin and its repair in mouse tissues including liver, kidney, and lung, genome-wide and at single- nucleotide resolution. We made the exciting discovery that for most genes the transcribed strand (TS) and non- transcribed strand (NTS) are repaired at different times of the day. We plan to take advantage of this finding to develop more efficient chronotherapy regimens, first for colorectal cancers, and in the future for other types of cancers that are treated with cisplatin and oxaliplatin. The proposed research is innovative because it is based on our discoveries in the fields of DNA repair and circadian clock, and it is significant because of its relevance for cancer prevention and treatment.

项目概要/摘要 我们致力于核苷酸切除修复和哺乳动物生物钟的分子机制。我们有 最近在这两个领域都取得了技术和机械方面的进步，并获得了直接的成果 这两个领域相互关联的证据。我们在这两个领域的发现都直接适用于人类 健康。我们将应用这些新进展来实现以下目标： (1) 我们开发了原始 XR-seq 方法的更高分辨率版本，用于全基因组单 通过核苷酸切除修复处理的所有 DNA 损伤修复的核苷酸分辨率图谱， 包括致癌物和化疗药物引起的损害。此外，我们还开发了 Damage-seq DNA 损伤的类似全基因组单核苷酸分辨率图谱的方法。我们已经使用了 结合两种方法来发现不与损坏热点重叠的修复热点和冷点 或冷点，并获得了有关基因组 3D 和修复的新信息。我们将继续描述这些 将修复与表观基因组标记、3D 基因组组织、染色质状态和复制时间联系起来的功能。 XR-seq 还使我们能够在果蝇和其他动物中发现一种新颖的转录偶联修复机制。 双翅目昆虫。我们将利用生化和遗传学的方法来解决这一机制 新颖的修复系统。 (2)我们将定义哺乳动物生物钟的分子机制。最近，我们展示了 隐花色素 (CRY)，而不是周期 (PER)，是哺乳动物转录-翻译的抑制因子 反馈环路（TTFL），并且 PER 可以作为抑制子或激活子，具体取决于特定的基因， 以 CRY 依赖的方式。我们将进行实验，在体外系统中重建该模型 纯化的蛋白质。 (3) 生物钟、癌症和化疗。我们第一次能够绘制这两种损害的地图 顺铂的形成及其在小鼠组织（包括肝、肾和肺）中的修复，全基因组和单 核苷酸分辨率。我们做出了令人兴奋的发现，对于大多数基因来说，转录链（TS）和非转录链 转录链（NTS）在一天中的不同时间进行修复。我们计划利用这一发现 开发更有效的时间疗法方案，首先针对结直肠癌，未来针对其他类型的癌症 用顺铂和奥沙利铂治疗的癌症。拟议的研究具有创新性，因为它基于 关于我们在 DNA 修复和生物钟领域的发现，由于其相关性而具有重要意义 用于癌症的预防和治疗。