DNA damage induced structural and dynamic changes at telomeres

我获得这个K99奖项的目标是获得额外的培训，成为一个独立的 在端粒蛋白-DNA和蛋白质-蛋白质相互作用的单分子研究领域的研究员。 这项研究将由Van Houten博士指导，Erie博士和Opresko博士赞助。这个 这三个实验室的专业知识和资源为我提供了一个极好的环境来提高我的技能 在AFM和单分子荧光成像、端粒蛋白生化和QPCR分析中。 我们假设，除了破坏TRF1、TRF2和POT1蛋白与DMA的结合外， 环境引起的端粒DNA损伤(如紫外线或氧化应激)可导致 端粒结合蛋白在DNA上随机不稳定的组装。这反过来又逐渐有利于 T-环结构的破坏和3‘端突出物的暴露。这项研究的具体目的是 两倍。第一个目标是评估环境引起的DMA损伤对G-四联体的影响 形成、蛋白质结合、蛋白质组装和T环形成。我们将使用AFM来检查 DMA损伤对G-四链组装的影响。大块DNA损伤对DNA结合的影响 POT1、TRF1和TRF2到短端粒DMA底物将使用凝胶迁移率进行评估 位移分析(EMSA)和T环的形成将使用AFM进行检查。在AFM研究中，加载 双链端粒DNA上的POT1(用量子点、量子点标记)将用作保护的报告 集合。第二个目的是评估DNA损伤对蛋白质-DNA动力学的影响 端粒DNA上的相互作用和蛋白质组装。我们将表征TRF1、TRF2、 利用原子力显微镜成像和EMSA对POT1-QD进行偶联。这些蛋白质-QD偶联物将用于 单分子荧光研究评价紫外线诱导的DNA损伤如何影响细胞动力学 TRF1-、TRF2-DNA相互作用和掩蔽素组装。众所周知，某些环境DNA 破坏性物质会导致端粒缩短。短端粒是各种人类的特征 疾病。这项研究将极大地促进我们对暴露在环境应激源中的理解。 如紫外线和氧化应激，与端粒功能障碍有关的病因有几个 人类疾病，包括与年龄相关的退行性疾病和癌症。

My goal in obtaining this K99 award is to acquire the additional training to become an independent investigator in the field of single-molecule studies of telomere protein-DNA and protein-protein interactions. This study will be under the guidance of Dr. Van Houten and sponsored by Drs. Erie and Opresko. The expertise and resources from these three labs provide an excellent environment for me to advance my skills in AFM and single-molecule fluorescence imaging, telomere protein biochemistry, and QPCR assays. We hypothesize that, in addition to disrupting TRF1, TRF2 and POT1 proteins binding to DMA, environmentally-induced DMA damage (such as UV light or oxidative stress) at telomeres can cause stochastically unstable assemblies of telomere binding proteins on DMA. This in turn progressively favors the disruption of the T-loop structure and the exposure of the 3' overhang. The specific aims of this study are two-fold. The first aim is to evaluate the effects of environmentally-induced DMA damage on G-quadruplex formation, protein binding, protein assemblies, and T-loop formation. We will use AFM to examine the effects of DMA damage on G-quadruplex assembly. The impact of bulky DMA lesions on the binding of POT1, TRF1 and TRF2 to short telomeric DMA substrates will be evaluated using electrophoresis mobility shift assays (EMSAs), and the T-loop formation will be examined using AFM. In AFM studies, loading of POT1 (marked by quantum dots, QDs) onto duplex telomeric DMA will be used as a reporter of shelterin assembly. The second aim is to evaluate the effects of DNA damage on dynamics of protein-DNA interaction and protein assembly on telomeric DNA. We will characterize the functionality of TRF1, TRF2, and POT1-QD conjugates using AFM imaging and EMSA. These protein-QD conjugates will be used in single-molecule fluorescence studies to evaluate how UV-induced DNA damage affects the dynamics of TRF1-, TRF2-DNA interactions and shelterin assembly. It is known that certain environmental DNA damaging agents cause increased telomere shortening. Short telomeres are characteristic of various human diseases. This study will greatly advance our understanding of how exposure to environmental stressors. such as UV light and oxidative stress, is associated with telomere dysfunction in the etiology of several human disorders including age-associated degenerative diseases and cancer.