Structure Function Analysis of Bacterial DNA Methyltransferase

赖希第1部分-技术建议的工作集中在细菌DNA甲基转移酶，M.EcoRI和M.BamHI，其分别甲基化GAATTC和GGATCC中下划线的N6和N4胞嘧啶。最近的研究显示，M.EcoRI能使DNA弯曲50度，并在甲基化之前翻转出腺嘌呤。 碱基翻转检测一种新的，动态检测，使用荧光碱基类似物，2-氨基嘌呤。 第一个目标是利用M.EcoRI和各种方法来研究碱基翻转和DNA弯曲过程。 预稳定状态荧光实验将用于确定碱基翻转是否是酶辅助的，或者酶是否简单地稳定了螺旋外碱基。 时间分辨荧光测量将用于确定酶-DNA复合物内螺旋外碱基的方向和迁移率。 将结合的DNA荧光测量值与蛋白质（色氨酸）荧光的类似变化进行比较，以便提供催化循环早期步骤的完整动力学描述。 该分析将提供有关酶介导的DNA构象变形的详细动力学信息。 通过与相关M.将研究TaqI酶以评估其功能作用。 第二个目标是确定M.EcoRI和M.使用X-射线衍射方法对BamHI进行鉴定。 已经获得了M.EcoRI-DNA复合物的小晶体和M.BamHI的晶体，它们的折射率为2.7埃。 提出了进一步的结晶试验。 提出了各种M.EcoRI突变体的结晶试验。 许多改变DNA非化学结构的酶，通过稳定DNA分子内的大规模构象变化来实现。 这些包括高达100度的显著DNA“弯曲”和DNA正常双螺旋之外的单个碱基的移动。 这些酶介导的DNA构象变化如何促进酶的功能尚不清楚。 该提案将使用细菌DNA甲基转移酶M. EcoRI来测量涉及弯曲和“碱基翻转”的DNA结构变化。 这种酶将甲基转移到GAATTC中的第二个腺嘌呤。 我们已经开发了一种新的基于荧光的测定来测量这种酶中的碱基翻转，并建议扩展这些实验来测量这种情况如何随着时间的推移而发生。 将进行相关实验，以测量一旦酶从双螺旋中移除，酶如何改变碱基的柔性和方向。 该提案还试图确定蛋白质中哪些氨基酸负责这些构象变化。 该提案的第二个长期目标是使用X射线晶体学方法确定酶的三维结构。

9603567 Reich Part 1-Technical The proposed work focuses on the bacterial DNA methyltransferases, M.EcoRI and M.BamHI, which methylate the underlined N6 and N4 cytosine in GAATTC and GGATCC, respectively. M.EcoRI was recently shown to bend DNA by 50 degrees and to flip out the adenine prior to methylation. Base-flipping was detected by a novel, dynamic assay which uses the fluorescent base analog, 2-aminopurine. The first goal uses M.EcoRI and various methods to investigate base-flipping and DNA bending processes. Presteady state fluorescence experiments will be used to determine if base-flipping is enzyme-assisted or if the enzyme simple stabilizes the extrahelical base. Time-resolved fluorescence measurements will be used to determine the orientation and mobility of the extrahelical base within the enzyme-DNA complex. The combined DNA fluorescence measurements will be compared to similar changes in protein (tryptophan) fluorescence so that a complete kinetic description of early steps in the catalytic cycle should be provided. This analysis will provide detailed kinetic information about the enzyme-mediated deformation of DNA conformation. Amino acid residues implicated in catalysis and adenine stabilization by structural homology to the related M. TaqI enzyme will investigated to assess their functional role (s). The second goal is to determine the structures of M.EcoRI and M. BamHI using X-ray diffraction methods. Small crystals of an M.EcoRI-DNA complex and crystals of M.BamHI which diffract to 2.7 angstrom have been obtained. Further crystallization trials are proposed. Crystallization trials of various M.EcoRI mutants are proposed. Part 2-Non technical Many enzymes which alter the ahemical structure of DNA, do so by stabilizing large scale conformational changes within the DNA molecule. These include significant DNA "bends" of up to 100 degrees and movement of individual bases outside of the normal double-helix of DNA. How these enzyme-mediated changes in DNA conf ormation contribute to the enzyme's function is not understood. This proposal will measure changes in DNA structure involving bending and "base flipping" using the bacterial DNA methyltransferase, M.EcoRI. This enzyme transfers a methyl group to the second adenine in GAATTC. We have developed a novel fluorescence-based assay to measure base-flipping in this enzyme, and propose to extend these experiments to measure how this occurs over time. Related experiments will be done to measure how the enzyme changes the flexibility and orientation of the base once it is removed from the double helix. The proposal also seeks to identify which amino acids within the protein are responsible for these conformational changes. A second long term goal of the proposal is to determine the three-dimensional structure of the enzyme using X-ray crystallographic methods.