Molecular Mechanism of Mammalian DNA Excision Repair and the Circadian Clock

摘要 我们致力于核苷酸切除修复的分子机制和哺乳动物 生物钟。我们最近在技术和机械两方面都取得了进步 并获得了这两个领域相互关联的直接证据 菲尔兹。我们在这两个领域的发现都直接适用于人类健康。我们会申请 这些新进展是为了实现以下目标： (1)我们开发了原始XR-SEQ方法的更高分辨率版本 基因组范围的单核苷酸分辨率图谱修复所有DNA损伤，即 由核苷酸切除修复处理，包括致癌物和 化疗药物。此外，我们还开发了类似的损伤序列方法。 DNA损伤的全基因组单核苷酸分辨图谱。我们已经使用了 两种方法相结合来发现修复热点和未修复的冷点 重叠损伤热点或冷点并在基因组3D上获得新信息 并进行修复。我们将继续描述这些特征，以将修复与表观基因组学联系起来 标记、3D基因组组织、染色质状态和复制时间。 (2)明确哺乳动物生物钟的分子机制。 最近，我们证明了隐色素(CRE)，而不是句点(PER)，是 哺乳动物转录-翻译反馈环(TTFL)中的抑制子，以及PER 在哭声中，根据特定基因的不同，扮演抑制者或激活者的角色 依赖的态度。我们将进行实验，在体外重建这个模型 系统中含有纯化的蛋白质。 (3)昼夜节律、癌症、化疗。这是我们第一次能够 为了绘制顺铂在包括肝脏在内的小鼠组织中的损伤形成和修复情况， 肾和肺，全基因组和单核苷酸分辨率。我们做了激动人心的 发现对于大多数基因，转录的链(TS)和非转录的链(TS) (NTS)在一天中的不同时间进行维修。我们计划利用这一发现 开发更有效的计时治疗方案，首先是针对结直肠癌，然后是 顺铂和奥沙利铂治疗的其他类型癌症的未来。这个 拟议的研究具有创新性，因为它基于我们在以下领域的发现 DNA修复和生物钟，它意义重大，因为它与癌症相关 预防和治疗。

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. (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.