Eco RI DNA Methyltransferase: Catalytic and Recognition Mechanisms

9412078 Reich几乎所有生物的DNA信息量都是通过腺嘌呤(N6)或胞嘧啶(C5和N4)的甲基化而扩大的。DNA甲基化参与控制组织特异性和发育调节的基因表达；保护宿主(细菌)DNA不受核酸内切酶的切割；DNA修复；衰老和遗传印记。建议对两种细菌DNA甲基转移酶进行研究：EcoRI N6腺嘌呤甲基转移酶和BamHI N4胞嘧啶甲基转移酶，前者使GAATTC中的第二个腺嘌呤甲基化，后者使GGATCC中的第一个胞嘧啶甲基化。现成的基因和大量的同质蛋白质为进行结构-功能分析提供了基础。总的目标是充分了解序列特异性DNA甲基化的分子细节，以设计新的生物催化剂，酶“模拟”，和/或修改目标酶的序列特异性。详细说明了两个具体目标。首先是确定EcoRI甲基转移酶或其底物的结构特征，其次是BamHI甲基转移酶的结构特征，这些结构特征对催化是必不可少的。第二个目的是通过对野生型蛋白以及点突变和缺失突变的X射线结晶学分析，确定EcoRI或BamHI甲基转移酶及其相应的蛋白质辅因子和蛋白质DNA辅因子复合体的三维结构。这些结构信息将有助于我们理解过去对EcoRI甲基转移酶结构和功能的研究，并为未来旨在阐明序列特异性DNA修饰的研究提供基础。%这些实验旨在研究细菌DNA甲基转移酶。这些酶通过甲基化腺嘌呤和胞嘧啶来修改独特的DNA序列。提出了用X射线结晶学和核磁共振对其结构进行表征的方法。此外，接触DNA的部分酶将使用激光交联法和质谱仪进行鉴定。功能鉴定将主要通过蛋白质工程和蛋白质修饰实验进行。这项研究的目标是从分子上了解这些酶是如何修改DNA中的独特位点的。序列特异性DNA修饰是一种普遍存在的生物学过程，在分子生物学领域有实际应用。***

9412078 Reich The information content of DNA is expanded in almost all organisms by methylation of adenine (N6) or cytosine (C5 and N4). DNA methylation is involved in the control of tissue specific and developmentally regulated gene expression; the protection of host (bacterial) DNA against endonuclease cleavage; DNA repair; aging; and genetic imprinting. The study of two bacterial DNA methyltransferases is proposed: EcoRI N6 adenine methyltransferase, which methylates the second adenine in GAATTC, and BamHI N4 cytosine methyltransferase, which methylates the first cytosine in GGATCC. The ready availability of the genes and large quantities of homogeneous protein provides the basis for the structure-function analyses to be performed. The general goal is to understand the molecular details of sequence-specific DNA methylation sufficiently to design novel biocatalysts, enzyme "mimics", and/or modify the sequence-specificity of the target enzyme. Two specific aims are detailed. The first is to identify structural features of the EcoRI methyltransferase or its substrate, and to a lesser extent the BamHI methyltransferase, that are essential for catalysis. The second aim is to determine the three-dimensional structure of the EcoRI or BamHI methyltransferase and their corresponding protein-cofactor as well as protein DNA-cofactor complexes using X-ray crystallographic analyses of the wild type proteins as well as point and deletion mutants. This structural information will aid our understanding of past structure-function studies with the EcoRI methyltransferase as well as provide the basis for future studies aimed at elucidating sequence-specific DNA modification. %%% These experiments are designed to characterize bacterial DNA methyltransferases. These enzymes modify unique DNA sequences by methylating adenine and cytosine. Structural characterization using X-ray crystallography and nuclear magnetic resonance is proposed. Also portions of the enzymes which contact the DNA will be identified using laser cross linking and mass spectrometry. Functional characterization will largely be through protein engineering and protein modification experiments. The goal of this research is to obtain a molecular understanding of how these enzymes modify unique sites within DNA. Sequence-specific DNA modification is a universal biological process which has practical applications in the field of molecular biology. ***