Biochemical Insight of Hyperphosphorylation of Replication Protein A

描述(申请人提供)：概述本项目的目标是阐明人类复制蛋白A(RPA)在DNA损伤反应中功能的生化基础，并更好地了解损伤诱导的突变和致癌。DNA损伤被广泛认为与人类癌症的诱发有关。人RPA是主要的单链DNA结合蛋白，在DNA复制、DNA修复、重组和DNA损伤检查点等DNA代谢途径中起着至关重要的作用。鉴于RPA在细胞对DNA损伤的反应中经历了过度磷酸化，推测该蛋白可能通过其RPA32亚基N末端的过度磷酸化在细胞DNA损伤反应中发挥调制作用。过度磷酸化是如何从生化机制上影响RPA的细胞活性的，目前尚不清楚。在本研究中，我们将使用一系列严格的生化方法：(1)确定RPA在DNA损伤诱导的过度磷酸化过程中的结构特征和结构特征；(2)表征过度磷酸化对RPA与具有生物学意义的DNA中间体相互作用的影响；以及(3)确定过度磷酸化诱导的RPA结构域相互作用的结合亲和力和热力学。这项拟议的研究与癌症生物学高度相关，因为人类DNA损伤反应中的缺陷会导致基因组不稳定，从而导致一系列具有高度癌症进化潜力的疾病。这个学术研究促进奖将允许本科生和研究生在医学院环境中获得生物医学研究经验，这将为他们在科学研究领域的职业生涯做好准备。具体地说，拟议的研究将解决一个重要的问题，即在细胞DNA损伤反应中RPA过度磷酸化的生化基础。这个项目与癌症生物学高度相关，因为DNA损伤是人类癌症的主要原因。

DESCRIPTION (provided by applicant): Summary The goal of this project is to elucidate the biochemical basis of the functions of human replication protein A (RPA) in DNA damage responses, and to better understand the damage-induced mutagenesis and carcinogenesis. DNA damage has been widely implicated in human cancer induction. Human RPA, the major single-stranded DNA binding protein, is essential for all DNA metabolic pathways such as DNA replication, DNA repair, recombination and DNA damage checkpoints. Given that RPA undergoes hyperphosphorylation in cells in response to DNA damage, it is hypothesized that the protein may play a modulatory role in cellular DNA damage responses mediated by the hyperphosphorylation at the N- terminus of its RPA32 subunit. How the hyperphosphorylation affect the cellular activities of RPA in terms of biochemical mechanism remains unknown. In this study, using an array of rigorous biochemical methods we will: (1) identify domain-domain interactions and structural characteristic of RPA upon DNA damage-induced hyperphosphorylation; (2) characterize the effects of hyperphosphorylation on RPA interaction with DNA intermediates of biological significance; and (3) determine binding affinity and thermodynamics of the hyperphosphorylation-induced domain-domain interaction of RPA. The proposed study is highly relevant to cancer biology as human defects in DNA damage responses lead to genome instability and thus a series of diseases with high potential to evolve cancer. This Academic Research Enhancement Award will allow undergraduate and graduate students to gain biomedical research experience in a medical school environment that will prepare them for careers in scientific research. Specifically, the proposed study will address an important question regarding the biochemical basis of RPA hyperphosphorylation in cellular DNA damage responses. This project is highly relevant to cancer biology as DNA damage is the major cause to human cancer.