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

项目总结 人们认为，包括癌症在内的许多疾病都是从DNA水平开始的。DNA是 不断受到内源性和外源性因素的轰炸，紫外线是其中之一 最强大的特工。细胞的防御机制是一种被称为DNA的酶 修复DNA损伤的聚合酶，被认为是基因组的守护者。其中 DNA聚合酶，DNA聚合酶Theta(POLQ)已被证明可以修复紫外线损伤 包括常见的顺式环丁烷-嘧啶二聚体(CPD)和a(6-4) 感光产品(PP)。尽管发挥了这个作用，POLQ在这种类型的修复过程中引入了突变，并且 目前尚不清楚它是保护了基因组，还是导致了基因组的不稳定。 该项目的目标是确定生物化学和机械基础 POLQ如何通过多种生化、生物物理和生物化学途径修复紫外线诱导的DNA损伤 细胞生物学方法。这项提案将检验两个关于癌症如何- 暴露在阳光下的肿瘤患者中POLQ的相关变体绕过了CPD损伤的DNA。 从这些实验中获得的关于需要绕过的生化机制的知识 紫外线损伤将为POLQ再次保护皮肤癌的想法提供支持证据 尽管有低水平的突变，但正常细胞和癌症相关变异是驱动因素 关于肿瘤发生的研究。这是第一次探索生物化学机制和 患者来源的POLQ突变的细胞表型。该项目的长期科学目标是 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，这项提议将确定 POLQ患者来源的变异(目标1)和确定核苷酸的机制 选择POLQ(目标2)。利用生化动力学和其他细胞学的初步研究 生物学技术表明，其中一种与癌症相关的变种经历了DNA改变 具有修复能力，对DNA损伤剂敏感。这些固有的区别在于 突变体和野生型POLQ为探索生化机制提供了机会 进一步扩大我们目前对细胞如何应对紫外线损伤的理解。 罗德岛学院的本科生研究人员将在这一成功中发挥不可或缺的作用 这个R15区的建议书。他们将参与项目的方方面面，包括 培养本科生的实验设计、数据解释和稿件准备 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.