Structure-Function Analysis of the EcoRI DNA Methylase

这一提议解决了生物化学中的一个基本问题-- 特定蛋白质-DNA相互作用的性质。 总目标是 为了阐明EcoRI甲基化酶如何催化 甲基从S-腺苷甲硫氨酸（蛋氨酸）到第二个 双链序列中的腺嘌呤：5'GAATTC 3'。 具体 目标是表征：（1）动力学机制，（2） 化学机制，（3）对特异性的贡献 来源于单个酶-底物相互作用，（4） 甲基化酶-DNA复合物的结构。 这些目标将以稳定和预先稳定的状态来实现 动力学、pH分析和修饰的DNA底物。 具体 氨基酸的化学修饰和遗传取代 将与动力学研究一起使用， 催化和底物结合中的残基。 的适用性 DNA足迹分析，以确定甲基化酶-DNA复合物 将测试拓扑结构和结合亲和力。 协同 晶体学和1H NMR。DNA甲基化酶分析 复杂的正在进行中。 DNA甲基化酶是体外操纵DNA甲基化酶的关键。 在基因表达的调控中起着关键作用 在真核生物中。 但没有结构和功能信息 可用于任何DNA甲基化酶。 拟议的工作旨在 在获得EcoRI甲基化酶的这一信息时， 这类重要的酶。

This proposal addresses a fundamental problem in biochemistry-the nature of specific protein-DNA interactions. The general goal is to elucidate how the EcoRI methylase catalyzes transfer of methyl group from S-adenosylmethionine (AdoMet) to the second adenine in the double stranded sequence: 5' GAATTC3'. Specific goals are to characterize: (1) the kinetic mechanism, (2) the chemical mechanism, (3) the contributions toward specificity deriving from individual enzyme-substrate interactions, (4) the structure of the methylase-DNA complex. These goals will be addressed with steady and presteady state kinetics, pH analyses, and modified DNA substrates. Specific chenical modification and genetic substitution of amino acids will be used with the kinetic studies to implicate specific residues in catalysis and substrate binding. The applicability of DNA-footprinting assays to determine the methylase-DNA complex topology and binding affinity will be tested. Collaborative crystallographic and 1H N.M.R. analyses of the methylase-DNA complex are ongoing. DNA methylases are essential for the in vitro manipulation of DNA and play a critical role in the regulation of gene expression in eukaryotes. Yet no structural and little function information is available for any DNA methylase. The proposed work is aimed at obtaining this information for the EcoRI methylase, a member of this important class of enzymes.