Structure and Function of Clustered DNA Lesions

描述（由申请人提供）：电离辐射和拟放射性化疗药物的一个独特性质是产生成簇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、SSB和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。除了DSB之外，由碱基和/或糖损伤和/或SSB组成的多重损伤位点（MDS）在低剂量电离辐射后容易在细胞中产生，并且它们占总簇状损伤的80%。最近的研究表明，修复这些MDS的尝试可能会产生不同的结果，这取决于损伤的类型、它们的分离和相对方向。使用纯化的DNA糖基化酶或核细胞提取物进行的DNA切割研究表明，一些MDS可以容易地切割，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。由相同病变构成的MDS可根据损伤分离和相对方向进行不同处理。目前，解释这种性质的结构基础几乎不存在。 在这个应用程序中，我们建议确定不同类型的集群双链病变的解决方案的结构，相关的结构与它们的识别纯化的DNA糖基化酶，并研究其处理细胞提取物。我们将使用高分辨率NMR光谱结合约束的分子动力学，以确定三维结构的DNA双链体含有集群双链损伤的损伤碱基（8-oxoG，DHT），脱碱基位点，或链断裂的组合，不同的损伤间的分离和方向。我们将使用纯化的DNA糖基化酶测定这些损伤的切割，以确定BER酶对它们的识别和加工。我们将使用真核细胞提取物测定上述MDS的修复潜力，以建立修复的程度和层次。完成这一建议将建立直接的相关性的解决方案的结构簇的DNA病变和它们的一些生物学特性，并将有助于了解电离辐射的毒性和致突变性的分子机制，一个知识与潜在的应用设计的新型化疗药物。

DESCRIPTION (provided by applicant): A unique property of ionizing radiation and radiomimetic chemotherapeutic drugs is the generation of clustered DNA damage, this is two or more DNA lesions (oxidized bases, modified sugars, SSB, and 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. In addition to DSB, multiply damaged sites (MDS) composed of base and/or sugar damage and/or SSB, are readily produced in the cell after low doses of ionizing radiation, and that they make up to 80% of the total clustered damage. It has been shown recently that, attempts to repair these MDS can produce different outcomes, depending on the type of damages, their separation, and relative orientation. DNA incision studies using purified DNA glycosylases 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. MDS made of identical lesions can be processed differently depending on damage separation and relative orientation. At the present time, the structural basis that explains this property is almost non-existent. In this application we propose to determine the solution structure of different types of clustered bistrand lesions, to correlate the structures with their recognition by purified DNA glycosylases, and to study their processing by cellular extracts. We will use high-resolution NMR spectroscopy in combination with restrained molecular dynamics to determine three-dimensional structures of DNA duplexes containing clustered bistrand lesions formed by a combination of damage bases (8-oxoG, DHT), abasic sites, or strand breaks, varying inter-lesion separation and orientation. We will assay cleavage of these lesions using purified DNA glycosylases to determine their recognition and processing by BER enzymes. We will assay the repair potential of the above-mentioned MDS using eukaryotic nuclear cell extracts to establish the extent and hierarchy of repair. Completion of this proposal will establish direct correlations between the solution structure of clustered DNA lesions and some of their biological properties, and will help to understand the molecular mechanisms of toxicity and mutagenicity of ionizing radiation, a knowledge with potential application for the design of novel chemotherapeutic drugs.