Structure Function Analysis of Bacterial DNA Methyltransferase

本文提出的工作重点是细菌DNA甲基转移酶M.EcoRI和m.b imhi，它们分别甲基化GAATTC和GGATCC中标记的N6和N4胞嘧啶。最近的研究表明，M.EcoRI可以使DNA弯曲50度，并在甲基化之前翻转腺嘌呤。碱基翻转是通过使用荧光碱基类似物2-氨基嘌呤的一种新的动态检测方法来检测的。第一个目标是使用m.e ecori和各种方法来研究碱基翻转和DNA弯曲过程。预稳态荧光实验将用于确定碱基翻转是酶辅助的还是酶简单地稳定螺旋外碱基。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。结合DNA荧光测量将与蛋白质（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。这一分析将提供详细的动力学信息，关于酶介导的DNA构象变形。与相关的M. TaqI酶具有结构同源性的催化和腺嘌呤稳定氨基酸残基将被研究以评估其功能作用。第二个目标是利用x射线衍射方法确定m.e ecori和m.b imhi的结构。获得了m.e ori - dna复合物的小晶体和m.b imhi晶体，其衍射范围为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化学结构的酶是通过稳定DNA分子内大规模的构象变化来实现的。这些包括显著的DNA“弯曲”，高达100度，以及DNA正常双螺旋结构外的单个碱基的移动。这些酶介导的DNA构象变化如何影响酶的功能尚不清楚。这项提议将使用细菌DNA甲基转移酶m.e ecori来测量DNA结构的变化，包括弯曲和“碱基翻转”。这种酶将甲基转移到GAATTC中的第二个腺嘌呤上。我们已经开发了一种新的基于荧光的测定方法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如何随时间发生。相关的实验将会进行，以测量酶如何改变碱基的灵活性和方向，一旦它从双螺旋结构中移除。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.