Repair of Radiation-Induced Crosslink Lesions of DNA

说明(申请人提供)：电离辐射和正常的有氧代谢都会导致活性氧物种(ROS)的形成，这可能会引起一系列DNA修饰，包括单核苷酸损伤、链断裂和交联性损伤。本应用的重点是了解未被探索的、辐射诱导的DNA链内和链间交联损伤的细胞识别和修复。建议的实验是根据三个具体目标组织的。在目标1中，我们将使用我们最新开发的穿梭载体方法来探索辐射诱导的链内交联损伤如何扰乱DNA转录，以及它们是如何在人类细胞中修复的。在目标2中，我们将研究辐射诱导的链间交联性损伤的形成以及NEIL1在电离辐射和其他因素引起的链内和链间DNA交联性损伤修复中的作用。在目标#3中，我们将使用定量蛋白质组学方法来鉴定能够与含有辐射诱导的交联性损伤的双链DNA特异结合的细胞蛋白质，并将评估这些新发现的蛋白质在修复哺乳动物细胞中的交联性损伤中的作用。这项研究的结果将为辐射诱导的链内和链间交联损伤的修复提供新的见解，并可能通过揭示DNA糖基酶NEIL1在修复这些损伤中的新作用以及通过发现作用于辐射诱导的交联损伤的新的损伤识别和DNA修复蛋白来改变我们对这类DNA损伤修复的理解。此外，所提出的蛋白质组学实验可能为鉴定与DNA损伤反应信号有关的新蛋白质提供依据。因此，这项拟议的研究将显著提高我们对暴露于外源性电离辐射对人类健康不利影响的理解，并最终可能导致增强型癌症放射治疗的发展。

DESCRIPTION (provided by applicant): Ionizing radiation and normal aerobic metabolism both lead to the formation of reactive oxygen species (ROS), which can give rise to a spectrum of DNA modifications including single-nucleobase lesions, strand breaks, and crosslink lesions. The emphasis of the present application is placed on understanding the cellular recognition and repair of the under-explored, radiation-induced intrastrand and interstrand crosslink lesions of DNA. The proposed experiments are organized according to three specific aims. In Aim #1, we will employ our newly developed shuttle vector method to explore how radiation-induced intrastrand crosslink lesions perturb DNA transcription and how they are repaired in human cells. In Aim #2, we will investigate the formation of radiation-induced interstrand crosslink lesions and the role of NEIL1 in repairing the intrastrand and interstrand DNA crosslink lesions induced by ionizing radiation and other agents. In Aim #3, we will employ a quantitative proteomic method to identify cellular proteins that are capable of binding specifically to duplex DNA harboring a radiation-induced crosslink lesion, and we will assess the roles of these newly identified proteins in repairing the crosslink lesions in mammalian cells. The outcome of the proposed research will provide novel insights into the repair of the radiation-induced intrastrand and interstrand crosslink lesions, and it may result in a paradigm shift in our understanding of the repair of this type of DNA lesions by revealing the novel role of DNA glycosylase NEIL1 in repairing these lesions and by discovering new damage recognition and DNA repair proteins acting on radiation-induced crosslink lesions. Additionally, the proposed proteomic experiments may afford the identification of novel proteins involved in DNA damage response signaling. Therefore, the proposed research will improve significantly our understanding of the adverse human health effects emanating from exposure to exogenous ionizing radiation and it may ultimately lead to the development of enhanced cancer radiotherapy.