Structure and Function of Clustered DNA Lesions

描述（由申请人提供）：电离辐射和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、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作用直接相关。除DSB外，由碱基和/或糖损伤和/或SSB组成的多重损伤位点（multiple damage sites， MDS）在低剂量电离辐射后很容易在细胞中产生，它们占总簇状损伤的80%。最近的研究表明，修复这些MDS的尝试可以产生不同的结果，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MDS切割得很差，在细胞中持续较长时间。由相同病变组成的MDS可以根据损伤的分离和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): 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.