Uncovering the Mechanistic Role of DNA Pol Theta in UV-Damage Repair

项目摘要 人们认为，包括癌症在内的许多疾病都是从DNA水平开始开始的。DNA是 不断受到内源性和外源性因素的轰击，紫外线是其中一种 最强大的代理商细胞的防御机制是一类称为DNA的酶 聚合酶修复DNA损伤，被认为是基因组的监护人。这些 DNA聚合酶，DNA聚合酶θ（POLQ）已被证明可以修复紫外线损伤 包括常见的病变顺式-顺式环丁烷-嘧啶二聚体（CPD）和a（6-4） 光产物（PP）。尽管有这种作用，POLQ在这种类型的修复过程中引入突变， 目前还不清楚它是保护基因组还是导致基因组不稳定。 本项目的目标是确定生物化学和机械基础， POLQ如何通过各种生物化学，生物物理学， 细胞生物学方法。这项提案将测试两个关于癌症如何- 来自阳光照射肿瘤患者的POLQ相关变体绕过CPD损伤的DNA。 从这些实验中获得的关于绕过 紫外线损伤将增加支持POLQ保护皮肤癌的证据。 正常细胞，尽管低水平的诱变和癌症相关的变异是驱动程序 肿瘤的发生。这是一项首创的研究，探索了生物化学机制， POLQ患者衍生突变体的细胞表型。长期科学目标 Towle-Weicksel研究小组将全面研究DNA的结构和功能 聚合酶影响基因组稳定性和致癌作用。 为了实现这些目标，本提案将确定 患者来源的POLQ变体（Aim 1），并确定核苷酸 选择POLQ（目标2）。利用生化动力学和其他细胞生物学方法的初步研究 生物学技术表明，其中一种癌症相关的变异经历了DNA的改变， 具有修复能力，对DNA损伤剂敏感。这些内在的差异， 变异和野生型POLQ提供了一个机会，探讨生化机制， 进一步扩展我们目前对细胞如何应对紫外线诱导损伤的理解。 来自罗得岛学院的本科研究人员将在成功中发挥不可或缺的作用 关于R15的建议他们将参与项目的各个方面，包括 实验设计，数据解释和手稿准备，以培养本科生 促进科学发现和培养科学技能的研究经验。

PROJECT SUMMARY It is thought that many diseases, including cancer, begin at the DNA level. DNA is constantly being bombarded by endogenous and exogenous factors, with UV rays as one of the most powerful agents. The cell's mechanism of defense is a class of enzymes known as DNA polymerases which repair DNA damage and are regarded as guardians of the genome. Of these DNA polymerases, DNA Polymerase Theta (POLQ) has been shown to repair UV damage including the common lesions cis-syn cyclobutane–pyrimidine dimer (CPD) and a (6-4) photoproduct (PP). Despite this role, POLQ introduces mutations during this type of repair, and it is unclear if it protects the genome or contributes to genomic instability. The objective of this project is to determine the biochemical and mechanistic basis for how POLQ repairs UV-induced DNA damage through a variety of biochemical, biophysical, and cellular biology approaches. This proposal will test two hypotheses about how cancer- associated variants of POLQ in patients from sun-exposed tumors bypass CPD-damaged DNA. The knowledge gained from these experiments on the biochemical mechanisms needed to bypass UV-damage will add supporting evidence to the idea that POLQ protects again skin cancer in normal cells despite low levels of mutagenesis and that the cancer-associated variants are drivers of tumorigenesis. This is a first-of-its-kind study that explores the biochemical mechanism and cellular phenotypes of patient-derived mutants of POLQ. The long-term scientific goal of the Towle-Weicksel research group is to fully investigate how structure and function of DNA polymerases influence genomic stability and carcinogenesis. To achieve these goals, this proposal will determine the catalytic mechanism of patient-derived variants of POLQ (Aim 1) and to determine the mechanism of nucleotide selection of POLQ (Aim 2). Preliminary studies using biochemical kinetics and other cellular biology techniques suggest that one of the cancer-associated variants experiences altered DNA repair abilities and is sensitive to DNA damaging agents. These inherent differences between the variant and wild-type POLQ provide an opportunity to explore the biochemical mechanism further to expand our current understanding of how the cell copes with UV-induced damage. Undergraduate researchers from Rhode Island College will play an integral role in the success of this R15 AREA proposal. They will be involved in all aspects of the project including experimental design, data interpretation, and manuscript preparation to foster an undergraduate research experience that advances scientific discovery and cultivates scientific skills.