SUBSTRATE RECOGNITION MECHANISM OF DNA REPAIR ENZYMES

该项目的长期目标是利用化学和机械方法来了解对人类健康和疾病有直接影响的生物系统。DNA携带着生命的遗传信息，编码方式是DNA分子长度上的碱基排列。不幸的是，这些碱在化学性质上也是活性的，因此很容易被修饰。自发的变化、内源性代谢物的损伤以及接触致癌物质等环境因素都会改变DNA的一级结构。维持基因组完整性的过程对生命的保真度至关重要。由DNA糖基酶启动的碱基切除修复通过从基因组中去除受损的碱基来帮助维持基因组的稳定性。为了发挥它们的功能，DNA糖基酶必须在大片未受损的DNA中定位受损的碱基底物。该项目的具体目标是确定DNA糖基酶在DNA中定位底物的机制。这种机制可能是进行性的，也可能是分配性的。一种过程性机制需要酶沿着DNA扩散，保持接触并扫描底物。一种分配机制涉及酶在三个维度扩散时的结合和解离。该计划是利用含有底物的DNA来测试底物识别的分布或过程模型。糖基酶活性将使用基于凝胶的分析来跟踪，圆二色谱将用于检测糖基酶或DNA的构象变化。当成功确定机制后，将通过研究模式生物(如大肠杆菌)中DNA糖基酶的特定突变来测试该模型的生物学相关性。了解确切的机制将导致了解DNA修复基因的哪些突变会导致功能变化，从而导致基因组稳定性的降低。

The long term goals of the project are to utilize chemical and mechanistic approaches to understand biological systems that have a direct impacct on human health and disease. DNA carries life's genetic information encoded in the arrangement of bases along the length of the DNA molecule. Unfortunately, these bases are also chemically reactive and therefore susceptible to modification. Spontaneous change, damage by endogenous metabolites, and exposure to environmental agents such as carcinogens can alter the primary structure of DNA. The processes that maintain genomic integrity are vitally important for the fidelity of life. Base excision repair initiated by DNA glycosylases helps to maintain genomic stability by removing damaged bases from the genome. In order to perform their function, the DNA glycosylases must locate damaged base substrates amongst vast tracts of undamaged DNA. The specific aims of the project are to determine the mechanism by which DNA glycosylases locate substrates in DNA. The mechanism might be either processive or distributive. A processive mechanism entails the diffusion of the enzyme along the DNA, maintiaining contact and scanning for substrates. A distributive mechanism involves the association and dissociation of the enzyme as it diffuses in three dimensions. The plan is to utilize substrate-containing DNA that is designed to test distributive or processive models for substrate recognition. Glycosylase activity will be followed using gel-based assays, and circular dichroism will be used to detect conformational changes in the glycosylase or the DNA. When the mechanism is successfully determined, the biological relevance of the model will be tested by studying specific mutants of DNA glycosylases in a model organism such as E. coli. Understanding precise mechanisms will lead to understanding which mutations in DNA repair genes cause alterations in function, and thus, lead to a decrease in genomic stability.