MECHANISM OF DNA REPAIR ENZYMES

因为DNA聚合酶β（Beta-pol）负责填补缺口， 在一些哺乳动物DNA修复途径中，它是最重要的合成途径之一。 维持基因组DNA完整性的重要酶。 B-pol 是药物设计的潜在目标，可以增强或阻断DNA， 修复过程。 然而，我们对分子机制的理解 B-pol还处于起步阶段，因此我们还没有足够的知识来开发 一个合理药物设计的程序。 为了弥补这一缺陷， 了解DNA核苷酸转移酶反应的基本原理 聚合酶，我们将集中在B-pol DNA合成的关键步骤 机理，酶-模板，引物结合。 该项目利用了 B-pol及其组成结构域在E.杆菌 包括：1）用X-射线衍射和透射电镜研究了B-pol的分子结构; 射线晶体学和多维NMR光谱学。 B-pol和 其结构域片段将结晶为与合成的 引物d（T）和其它合成模板引物。 NMR分析将 B-pol片段的分子量范围为-6至12- kDa，代表折叠的 完整蛋白质中的蛋白酶抗性结构域。 获得的结构 通过这些方法将检查对功能的影响， 分子建模和定点诱变，然后进行功能性 突变蛋白的测定; 2）B-pol功能的研究，如结合 模板引物和引物底物：这些将使用平衡 结合和酶学技术，包括预稳定动力学。 的 酶模板引物结合口袋，通过光化学交叉定位， 链接和结构研究，将被改变的网站定向 诱变 通过B-pol的复制研究将寻找 B-pol. Frameshaft DNA合成产物的序列变异性 突变热点占变异的大部分， 可能是由于模板引发的错误。引物滑动 机制等 我们将研究体外产生的突变，以确定 模板.引物-B-pol相互作用在模板.引物 滑动针对B-pol的药物设计的一个目标是开发药物 可以通过抑制DNA修复来增强化疗效果。 从盖普开始- 在许多类型的DNA损伤的修复过程中需要填充合成， B-pol是药物干预的合理选择。 药物的第二个目标 设计是通过寻找增加DNA修复的试剂来增强DNA修复。 活动和/或B-pol的准确性。

Because DNA polymerase beta (Beta-pol) is responsible for gap-filling synthesis in some mammalian DNA repair pathways, it is one of the most important enzymes for maintaining the integrity of genomic DNA. B-pol is a potential target for drug design to either enhance or block the DNA repair process. However, our understanding of the molecular mechanisms of B-pol is in its infancy, so that we do not yet know enough to develop a program of rational drug design. To correct this deficiency, and to understand basic principles of the nucleotidyltransferase reaction of DNA polymerases, we will focus on a key step in the B-pol DNA synthesis mechanism, enzyme-template.primer binding. The project exploits recombinant expression of B-pol and its constituent domains in E. coli and involves: 1) Studies of the molecular structure of B-pol both by X- ray crystallography and by multidimensional NMR spectroscopy. B-pol and its domain fragments will be crystallized as complexes with the synthetic primer d(T) and with other synthetic template primers. NMR analysis will be with B-pol fragments ranging from -6 to 12- kDa, representing folded protease-resistant domains in the intact protein. Structures obtained by these approaches will be examined for implications on function by molecular modeling and site-directed mutagenesis, followed by functional assays of mutant proteins; 2) Studies of B-pol functions, such as binding to template primer and primer substrates: These will use equilibrium binding and enzymological techniques, including pre-steady kinetics. The enzyme-template.primer binding pocket, localized by photochemical cross- linking and structural studies, will be altered by site-directed mutagenesis. Studies of replication by B-pol will seek the cause of sequence variability among products of DNA synthesis by B-pol. Frameshaft mutational hot spots account for much of the variability, a process probably due to mistakes that are initiated by template.primer slippage mechanisms. We will study mutations produced in vitro to determine if template.primer-B-pol interactions play a role in the template.primer slippage. One goal of drug design targeted to B-pol is to develop agents that can potentiate chemotherapy by inhibiting DNA repair. Since gap- filling synthesis is required during repair of many types of DNA lesions, B-pol is a logical choice for drug intervention. A second goal of drug design is enhancing DNA repair by finding agents that increase the activity and/or accuracy of B-pol.