Structure and Function of Clustered DNA Lesions

描述(申请人提供)：电离辐射和拟放射治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更多DNA损伤(氧化碱基、修饰糖、SSB和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。除DSB外，在低剂量电离辐射下，细胞内很容易产生由碱基和/或糖损伤和/或SSB组成的多重损伤位点(MDS)，它们占总簇状损伤的80%。最近已经表明，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糖基酶或核细胞提取物进行的DNA切割研究表明，一些MDS很容易被切割，产生有毒的DSB，而另一些切割得很差，在细胞中持续更长的时间。由相同的损伤构成的MDS可以根据损伤分离和相对方向的不同进行不同的处理。目前，解释这一性质的结构基础几乎不存在。 在这一应用中，我们建议确定不同类型聚集性双链病变的溶液结构，将结构与纯化的DNA糖基酶识别相关联，并研究细胞提取物对它们的处理。我们将使用高分辨率核磁共振光谱结合约束分子动力学来确定包含由损伤碱基(8-oxoG，DHT)、基本位置或链断裂的组合形成的簇状双链损伤的DNA双链的三维结构，这些损伤之间的分离和方向不同。我们将使用纯化的DNA糖基酶来检测这些损伤的切割，以确定BER酶对它们的识别和处理。我们将使用真核细胞提取物检测上述MDS的修复潜力，以确定修复的程度和层次。这一提议的完成将在簇状DNA损伤的溶液结构和它们的一些生物学特性之间建立直接的关联，并将有助于理解电离辐射毒性和诱变性的分子机制，这一知识在设计新型化疗药物方面具有潜在的应用前景。

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.