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·桑托斯博士申请过渡职业发展奖，他是纽约州立大学石溪分校药学系的一名初级教员。de los Santos博士是DNA损伤领域的结构生物学家，去年获得了NCI颁发的职业发展奖（K01）。申请人的长期职业目标是在分子水平上了解DNA损伤的识别和修复过程，并将这些过程与化学和环境诱变和致癌联系起来。目前的应用涉及DNA中多重损伤位点（multiple damage Sites， MDS）的研究。电离辐射和一些模拟放射化疗药物的一个独特特性是产生簇状DNA损伤，这是位于DNA螺旋单转内的两个或多个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作用直接相关。最近的研究表明，除了DSB外，由碱和/或糖损伤组成的MDS在低剂量电离辐射后也很容易在细胞中产生，并且它们占总簇状损伤的80%。修复MDS的尝试可以产生不同的结果，这取决于病变的类型、分离程度和相对方向。使用纯化酶或核细胞提取物进行的糖基酶活性研究表明，一些MDS很容易被切割，产生有毒的DSB，而另一些MDS切割得很差，在细胞中持续较长时间。此外，由相同病变组成的MDS可以根据损伤的分离和/或相对方向进行不同的处理。目前，解释这一特性的结构基础几乎不存在。

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.