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: 10458623
• 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/10458623
• 关键词: 3-Dimensional; Area; Biochemical Genetics; Carcinogens; Chemotherapy-Oncologic Procedure; 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; Proteins; Regimen; Regulation; Research; Resolution; System; Time; Tissues; Transcription-Coupled Repair; Translations; Work; base; cancer prevention; cancer therapy; cancer type; circadian pacemaker; cryptochrome; epigenomics; experimental study; genetic approach; genome-wide; innovation; novel; oxaliplatin; 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.

项目摘要/摘要 我们研究核苷酸切除修复和哺乳动物昼夜节律时钟的分子机制。我们有 最近在这两个领域都取得了技术和机械进步，并获得了直接 这两个领域相互联系的证据。我们在这两个领域的发现直接适用于人类 健康。我们将把这些新的进步应用于以下目标： （1）我们开发了针对全基因组单XR-seq方法的较高分辨率版本 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映射， 包括致癌物和化学治疗药物引起的损害。此外，我们开发了损坏序言 与DNA损伤的类似基因组单核苷酸分辨率映射的类似方法的方法。我们已经使用了 发现不重叠损害热点的两种方法的方法的结合 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机联系起来的功能。 XR-Seq还使我们能够发现果蝇和其他的新型转录耦合修复机制 昆虫以双翅目为单位。我们将使用生化和遗传方法来解决此机制 新颖的维修系统。 （2）我们将定义哺乳动物昼夜节律时钟的分子机制。最近，我们证明了 该加密色素（哭泣），而不是周期（per），是哺乳动物转录翻译中的阻遏物 反馈循环（TTFL），并且根据特定基因的不同，每行为作为阻遏物或激活剂 以哭泣的方式。我们将进行实验，以在体外系统中重新构建此模型 纯化的蛋白质。 （3）昼夜节律时钟，癌症和化学疗法。我们第一次能够绘制两个损坏 顺铂形成及其在包括肝，肾脏和肺在内的小鼠组织中的修复，全基因组以及单个 核苷酸分辨率。我们提出了一个令人兴奋的发现，即对于大多数基因而言，转录的链（TS）和非 - 在一天中的不同时间修复转录的链（NTS）。我们计划利用这一发现 开发更有效的年度疗法方案，首先是结直肠癌，将来用于其他类型 用顺铂和奥沙利铂治疗的癌症。拟议的研究具有创新性，因为它是基于的 关于我们在DNA修复和昼夜节律钟的发现，这很重要，因为它的相关性 用于预防癌症和治疗。