SEQUENCE-SPECIFIC DNA-PROTEIN INTERACTIONS

我们研究的主要长期目标是了解 蛋白质识别并与特定分子相互作用的分子机制 DNA序列 这个问题正在通过一个详细的 II型限制和修饰结构和功能研究 系统. EcoRI系统的特征最好。 基因和氨基酸 这两种酶的酸序列已经确定，和X射线 晶体学分析开始提供关于 核酸内切酶-DNA复合物的结构。 互补触点 将探索酶与其底物序列之间的关系。 EcoRI内切酶的突变体将被分离和定位。 改变 酶将被纯化并分析功能参数， DNA水解动力学，二聚体结构稳定性，平衡 结合常数和与DNA底物的接触。 其它II型 系统将被检查，以确定的一般性， 来源于EcoRI系统。 特别地，异构体 在红球藻中鉴定出了EcoRI酶切位点。 虽然R. sphaeroides和E.大肠杆菌是相当远的关系，初步 实验表明这些核酸内切酶具有共同的起源。 RsrI 限制性酶切和修饰系统克隆到E.杆菌 DNA 将使用序列分析来推断所述蛋白质的氨基酸序列。 内切酶 酶将被纯化，功能参数将被确定。 与EcoRI的同行相比。 这两个数据的比较 系统将分析与三维结构的关系， 通过X-射线晶体学分析得到的野生型酶-DNA复合物 分析。 识别DNA中的特定序列是一个重要的过程 在DNA复制，重组和基因组的表达过程中， 细胞代谢和发育。 阐明参与的机制 利用相对简单的系统的序列特异性相互作用应该 阐明这些复杂的过程。 II型限制性修饰所利用的基因调控模式 系统尚未针对任何系统阐明。 在EcoRI的案例中， 系统，已经表明，甲基化酶是需要激活 内切核酸酶的表达。 这种激活的机制还没有 被识别。 β-半乳糖苷酶融合质粒将用于 监测甲基化酶活化。 体内转录将通过以下方法分析： 北方印迹分析和S1定位。

The major long-term objective of our research is to understand the detailed molecular mechanisms by which proteins recognize and interact with specific DNA sequences. This problem is being approached through a detailed structural and functional study of type II restriction and modification systems. The EcoRI system is the best characterized. The gene and amino acid sequences of both enzymes have been determined, and X-ray crystallographic analysis is beginning to yield detailed information about the structure of the endonuclease-DNA complex. Complementary contacts between the enzymes and their substrate sequence will be explored. Mutants of the EcoRI endonuclease will be isolated and mapped. Altered enzymes will be purified and analyzed for functional parameters such as kinetics of DNA hydrolysis, stability of dimer structure, equilibrium binding constants and contacts with the DNA substrate. Other type II systems will be examined in order to determine the generality of information derived from the EcoRI system. In particular, an isoschizomer of EcoRI has been identified in Rhodopseudomonas sphaeroides. Although R. sphaeroides and E. coli are rather distantly related, preliminary experiments indicate that the endonucleases have a common origin. The RsrI restriction and modification system will be cloned into E. coli. DNA sequence analysis will be used to deduce the amino acid sequences of the enzymes. The enzymes will be purified and functional parameters will be compared to the EcoRI counterparts. Comparative data from both of these systems will be analyzed in relation to the three-dimensional structure of the wild type enzyme-DNA complex derived by X-ray crystallographic analyses. Recognition of defined sequences in DNA is an essential process in DNA replication, recombination and the expression of the genome during cellular metabolism and development. Elucidation of mechanisms involved in sequence-specific interactions utilizing relatively simple systems should illuminate these complex processes. The mode of gene regulation utilized by type II restriction-modification systems has not been elucidated for any system. In the case of the EcoRI system, it has been shown that the methylase is required to activate ezpression of the endonuclease. The mechanism of this activation has not been identified. Beta-galactosidase fusion plasmids will be used to monitor methylase activation. In vivo transcription will be analyzed by Northern blot analysis and S1 mapping.