Recognition of Multiply Damaged Sites in DNA

描述（由申请人提供）： 本申请是为卡洛斯德洛斯桑托斯博士申请的过渡职业发展奖，他是纽约州立大学斯托尼布鲁克分校药理科学系的初级教员。 德洛斯桑托斯博士是一位结构生物学家，致力于DNA损伤领域的研究，并在NCI职业发展奖（K01）的最后一年。 申请人的长期职业目标是了解在分子水平上识别和修复DNA损伤的过程，并将这些过程与化学和环境诱变和致癌作用相关联。 本申请涉及DNA中多重损伤位点（MDS）的研究。 电离辐射和一些拟放射性化疗药物的独特性质是产生成簇的DNA损伤，这是位于DNA螺旋的单圈内的两个或更多个DNA损伤（氧化碱基、修饰糖、单链（SSB）和双链断裂（DSB））。 一段时间以来，人们已经知道DSB的数量与电离辐射的杀伤效应直接相关。 最近，已经表明，除了DSB之外，在低剂量的电离辐射后，细胞中容易产生由碱基和/或糖损伤组成的MDS，并且它们占总簇状损伤的80%。 试图修复MDS可能会产生不同的结果，这取决于病变的类型，它们的分离和相对方向。 使用纯化的酶或核细胞提取物进行的糖基化酶活性研究表明，一些MDS可以容易地被切割，产生毒性DSB，而其他MDS切割非常差，在细胞中持续更长的时间。 此外，由相同病变组成的MDS可以根据损伤分离和/或相对取向进行不同的处理。 目前，解释这种性质的结构基础几乎不存在。 我们目前正在使用高分辨率NMR光谱和约束的分子动力学模拟，以确定三维结构的DNA双链体含有代表性的MDS。 为了将结构与生物功能相关联，我们建议确定模型簇状病变的识别和修复特性。 我们将研究在核细胞提取物的存在下，通过纯化的BER蛋白对MDS的识别和处理。 我们将使用真核细胞提取物研究MDS的碱基切除修复，以建立修复的程度和层次。 我们将分离结合这些病变的核细胞蛋白，并通过质谱方法确定其身份。 完成这一提议将建立成簇双链病变的溶液结构与其某些生物学特性之间的关系。 此外，它将为申请人提供更多的时间，以便在其实验室建立独立的研究计划。

DESCRIPTION (provided by applicant): This application is for a Transition Career Development Award for Dr. Carlos de los Santos, who is a junior faculty member in the Department of Pharmacological Sciences at SUNY Stoney Brook. Dr. de los Santos is a structural biologist working in the field of DNA damage and is in the last year of Career Development Award (K01) from the NCI. The applicant's long-term career goal is to understand processes involved in the recognition and repair of DNA lesions at the molecular level, and to correlate these processes with chemical and environmental mutagenesis and carcinogenesis. The present application involves the study of Multiply Damaged Sites (MDS) in DNA. A unique property of ionizing radiation and some radiomimetic chemotherapeutic drugs is the production of clustered DNA damage, this is two or more DNA lesions (oxidized bases, modified sugars, single (SSB) and double strand breaks (DSB)) located within a single turn of the DNA helix. It has been known for some time that the number of DSB correlates directly with the kill effects of ionizing radiation. Recently, it has been shown that, in addition to DSB, MDS composed of base and/or sugar damages are readily produced in the cell after low doses of ionizing radiation, and that they make up to 80% of the total clustered damage. Attempts to repair MDS can produce different outcomes, depending on the type of lesions, their separation, and relative orientation. Glycosylase activity studies using purified enzymes or nuclear cell extracts showed that some MDS can be cleaved readily generating toxic DSB, while others are incised very poorly, persisting in the cell for longer periods of time. Furthermore, MDS composed by identical lesions can be processed differently depending on damage separation and/or relative orientation. At the present time, the structural basis that explains this property is almost non-existent. We are currently using high-resolution NMR spectroscopy and restrained molecular dynamics simulations to determine three-dimensional structures of DNA duplexes containing representative MDS. In order to correlate the structures with biological function, we propose to determine recognition and repair properties of model clustered lesions. We will investigate recognition and processing of MDS by purified BER proteins and in the presence of nuclear cell extracts. We will examine base excision repair of MDS using eukaryotic nuclear cell extracts to establish the extent and hierarchy of repair. We will isolate nuclear cell proteins that bind these lesions and establish their identity by mass spectroscopy methods. Completion of this proposal would establish relationships between the solution structure of clustered bistrand lesions and some of their biological properties. Additionally, it would afford additional time to the applicant for the establishment of an independent research program in his laboratory.