STRUCTURAL BASIS FOR THE SUBSTRATE SPECIFICITY OF THE BER ENZYMES

该项目的长期目标是了解在电离辐射修复中起关键作用的两个酶家族的底物特异性的结构基础：HhH-GPD Nth超家族和Fpg家族。所提出的工作的基本假设是，任何糖基化酶超家族或家族成员提供的分子支架可以支持各种各样的特异性切除后的碱基取代少量的残基。 具体的目标如下：1-我们将解决的DNA结合的复合物的DNA糖基化酶选择的系统发育/结构算法在核心A。我们将研究第N超家族成员的一个子集，其中糖基化酶中的氨基酸取代导致底物特异性改变。同样，我们将研究复合物的一个子集的天然存在的DNA糖基化酶，具有相似的序列，但不同的底物特异性，如EcNth和微球菌luteus紫外核酸内切酶（M1 Uve）。2-我们还将解析Fpg家族成员的一个子集的DNA结合复合物的晶体结构，所述Fpg家族成员包括Fpg和Nei蛋白的天然人直系同源物，人NEI 1 L， NEIL 2和NEIL 3与它们的DNA底物复合。此外，我们将比较NEIL 1与拟南芥Fpg的配体结构，因为这两种酶具有序列相似性，但识别非常不同的底物。 我们的结构研究将补充现有的结构数据和项目1中的底物特异性研究，并将它们结合在一起，为更好地理解这些类别的DNA糖基化酶的底物特异性提供良好的结构基础。

The long-term goal of this project is to understand the structural basis for substrate specificity in two families of enzymes that play a critical role in the repair of ionizing radiation: the HhH-GPD Nth Superfamily and the Fpg Family. The fundamental hypothesis underlying the proposed work is that the molecular scaffold provided by any glycosylase superfamily or family member can support a wide variety of specificities for the excised base after substitution of a small number of residues. The specific aims are as follows: 1- We will solve the crystal structures of DNA-bound complexes of DNA glycosylases selected by phylogenetic/structural algorithms developed in Core A. We will study a subset of the Nth superfamily members where amino acid substitutions in the glycosylase result in altered substrate specificity. Similarly, we will examine complexes of a subset of naturally occurring DNA glycosylases that have similar sequences but divergent substrate specificities, such as EcNth and Micrococcus luteus UV endonuclease (M1Uve). 2- We will also solve the crystal structures of the DNA bound complexes of a subset of the Fpg Family members including the natural human orthologs of Fpg and Nei proteins, human NEI1 L, NEIL2, and NEIL3, in complex with their DNA substrate(s). Additionally, we will compare the liganded structm'e of NEIL1 with that ofArabidopsis thaliana Fpg, as the two enzymes share sequence similarity, but recognize very different substrates. Our structural studies will complement the available structural data and the substrate specificity studies in Project 1 and taken together they should provide a sound structural basis for a better understanding of substrate specificity in these classes of DNA glycosylases.