Structure Function Analysis of Bacterial DNA Methyltransferase

9603567德国第1部分-技术拟议的工作重点是细菌DNA甲基转移酶M.EcoRI和M.BamHI，它们分别使GATTC和GGATCC中下划线的N6和N4胞嘧啶甲基化。最近的研究表明，M.EcoRI可以将DNA弯曲50度，并在甲基化之前将腺嘌呤翻转出来。碱基翻转是通过一种新的、动态的检测方法来检测的，该方法使用了荧光碱基类似物2-氨基嘌呤。第一个目标是使用M.EcoRI和各种方法来研究碱基翻转和DNA弯曲过程。稳定前的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的碱基。时间分辨荧光测量将用于确定酶-DNA复合体中螺旋外碱基的取向和迁移率。联合的DNA荧光测量将与蛋白质(色氨酸)荧光的类似变化进行比较，因此应该提供催化循环早期步骤的完整动力学描述。这一分析将提供有关酶介导的DNA构象变形的详细动力学信息。通过与相关的分枝杆菌Taqi酶的结构同源性，研究与催化和腺嘌呤稳定有关的氨基酸残基，以评估它们的功能作用(S)。第二个目标是用X射线衍射法确定M.EcoRI和M.BamHI的结构。获得了微米级EcoRI-DNA复合体的小晶体和微米级BamHI的2.7埃衍射晶体。提出了进一步的结晶实验。对不同的M.EcoRI突变体进行了结晶试验。第2部分-非技术性的许多酶通过稳定DNA分子内的大规模构象变化来改变DNA的非化学结构。其中包括高达100度的显著DNA“弯曲”，以及单个碱基在正常的DNA双螺旋之外的移动。这些酶介导的DNA构象变化如何有助于酶的功能尚不清楚。这项建议将使用细菌DNA甲基转移酶M.EcoRI来测量DNA结构的变化，包括弯曲和“碱基翻转”。这种酶将一个甲基转移到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分析来测量这种酶的碱基翻转，并建议扩展这些实验来测量这种情况是如何随着时间的推移发生的。将进行相关实验，以测量一旦从双螺旋中移除，该酶将如何改变碱基的灵活性和取向。该提案还试图确定蛋白质中的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射线结晶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.