Recognition of Multiply Damaged Sites in DNA

描述(由申请人提供)：本申请是为纽约州立大学斯通尼·布鲁克分校药理科学系初级教员卡洛斯·德·洛斯桑托斯博士颁发的过渡职业发展奖。德洛斯桑托斯博士是一位在DNA损伤领域工作的结构生物学家，是NCI职业发展奖(K01)的最后一年。申请者的长期职业目标是在分子水平上了解识别和修复DNA损伤的过程，并将这些过程与化学和环境突变和致癌联系起来。目前的应用涉及DNA中多个损伤位点(MDS)的研究。电离辐射和一些仿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螺旋单圈内的两个或多个DNA损伤(氧化碱基、修饰糖、单链(SSB)和双链断裂(DSB))。DSB的数量与电离辐射的杀伤效应直接相关，这一点已经有一段时间了。最近的研究表明，除DSB外，在低剂量电离辐射下，细胞内容易产生由碱基和/或糖损伤组成的MDS，并且它们占总簇状损伤的80%。修复MDS的尝试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，在细胞中持续更长的时间。此外，由相同的损伤组成的MDS可以根据损伤分离和/或相对方向进行不同的处理。目前，解释这一性质的结构基础几乎不存在。 我们目前正在使用高分辨率核磁共振波谱和受限分子动力学模拟来确定含有代表性MDS的DNA双链的三维结构。为了将这些结构与生物功能联系起来，我们建议确定模型聚集性病变的识别和修复特性。我们将研究纯化的BER蛋白和核细胞提取物对MDS的识别和处理。我们将使用真核细胞提取物检测MDS的碱基切除修复，以确定修复的程度和层次。我们将分离结合这些损伤的核细胞蛋白，并通过质谱学方法确定它们的身份。 这一提议的完成将建立聚集性双链病变的溶液结构与其一些生物学特性之间的关系。此外，这将使申请人有更多的时间在其实验室建立一个独立的研究方案。

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.