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，这些信息编码在沿DNA分子长度排列的碱基中。不幸的是，这些碱基也具有化学反应性，因此容易被修饰。自发变化、内源性代谢物损伤以及暴露于环境因素（如致癌物）可改变DNA的初级结构。维持基因组完整性的过程对生命的保真度至关重要。碱基切除修复由DNA糖基酶启动，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。为了发挥其功能，DNA糖基酶必须在大量未受损的DNA中定位受损的碱基底物。该项目的具体目的是确定DNA糖基酶在DNA中定位底物的机制。这种机制可能是渐进的，也可能是分配的。过程机制需要酶沿着DNA扩散，保持接触并扫描底物。分配机制涉及酶在三维空间扩散时的结合和解离。该计划是利用含有底物的DNA，设计用于测试底物识别的分布或过程模型。糖基酶的活性将采用凝胶为基础的测定，而圆二色性将用于检测糖基酶或DNA的构象变化。当机制被成功确定后，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。了解精确的机制将有助于了解DNA修复基因中的哪些突变导致功能改变，从而导致基因组稳定性下降。

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.