Analysis of DNA Sequence Recognition by Three Types of Sequence-Specific Protein

这个项目的长期目标是了解如何 蛋白质识别特定的DNA序列， 机构是不同的，以适应功能的要求， 蛋白质。 为此，突变体特征的组合 和蛋白质生物化学将被用来研究三个功能 不同的DNA结合蛋白都来自Pvu II 限制-修改系统。 第一种蛋白质是 结合S-腺苷甲硫氨酸的Pvu II DNA甲基转移酶 和DNA双链体序列CAGCTG，并甲基化氨基 内部胞嘧啶。 第二种蛋白质是Pvu II 限制性内切酶，其结合相同的DNA序列， Mg ++和催化磷酸二酯裂解之间的中心两个 基地 第三种蛋白质是Pvu II C蛋白，它似乎 在DNA上具有更简并的识别特异性， 激活核酸内切酶基因的转录。 立即 目标是确定起关键作用的结构域和氨基酸 在识别底物DNA序列中的作用， 基板也是。 了解序列特异性识别 DNA被蛋白质，特别是当催化中心必须 与DNA接近，是一个重要的目标， 对基因表达的深刻理解， 生物技术的进一步发展。 %%% 这个项目的两个长期目标是了解如何 蛋白质识别特定的DNA碱基序列， 细胞中过量的非底物DNA，以及这些序列 识别机制不同，以适应其他功能 当催化中心必须被引入时， 靠近DNA进行反应。 了解序列- 蛋白质对DNA的特异性识别是一个重要的目标， 为了充分理解基因表达的控制， 生物技术的进一步发展。 在这项研究中，结合 诱变和蛋白质生物化学将被用来研究三个 DNA结合蛋白都来自Pvu II限制性酶 这两个系统有不同的功能，但每个系统都有不同的功能。 这些蛋白质中的第一种是Pvu II DNA甲基转移酶， 其结合S-腺苷甲硫氨酸和DNA双链体序列CAGCTG 并甲基化内部胞嘧啶的氨基。 的 第二种蛋白质是Pvu II限制性内切酶，其结合 相同DNA序列具有Mg ++并催化磷酸二酯裂解 中间两个基地。 第三种蛋白质是Pvu II C 蛋白质，它似乎结合Pvu II DNA并激活 核酸内切酶基因的转录。 近期目标是 确定这三种蛋白质的结构域和氨基酸， 在识别底物DNA序列方面发挥关键作用， 识别其他底物，并在进行化学反应时， 甲基化或裂解反应。

The long-term goal of this project is to understand both how proteins recognize specific DNA sequences, and how the recognition mechanisms are varied to accommodate the functional requirements of the protein. To that end, a combination of mutant characterization and protein biochemistry will be used to study three functionally disparate DNA-binding proteins which all come from the Pvu II restriction-modification system. The first of these proteins is the Pvu II DNA methyltransferase, which binds S-adenosylmethionine and the DNA duplex sequence CAGCTG and methylates the amino group of the internal cytosine. The second protein is the Pvu II restriction endonuclease, which binds the same DNA sequence and Mg++ and catalyzes phosphodiester cleavage between the central two bases. The third protein is the Pvu II C protein, which appears to have a more degenerate recognition specificity on the DNA and activates transcription of the endonuclease gene. The immediate goals are to identify the domains and amino acids that play key roles in recognizing the substrate DNA sequence and the other substrates as well. Understanding sequence-specific recognition of DNA by proteins, particularly when a catalytic center must be brought into proximity with the DNA, is an important goal both for a sophisticated understanding of gene expression and for the further development of biotechnology. %%% The two long-term goals of this project are to understand how proteins recognize specific DNA base sequences despite the large excess of nonsubstrate DNA in the cell, and how these sequence recognition mechanisms vary to accommodate the other functional requirements of the protein when a catalytic center must be brought close to the DNA to carry out reactions. Understanding sequence- specific recognition of DNA by proteins is an important goal both for fully understanding the control of gene expression, and for the further development of biotechnology. In this study, a combination of mutagenesis and protein biochemistry will be used to study three DNA-binding proteins which all come from the Pvu II restriction- modification system but which each have very different fuctions. The first of these proteins is the Pvu II DNA methlytransferase, which binds S-adenosylmethionine and the DNA duplex sequence CAGCTG and methylates the amino group of the internal cytosine. The second protein is the Pvu II restriction endonuclease, which binds the same DNA sequence an Mg++ and catalyzes phosphodiester cleavage between the central two bases. The third protein is the Pvu II C protein, which appears to bind the Pvu II DNA and activate transcription of the endonuclease gene. The immediate goals are to identify the domains and amino acids of these three proteins that play key roles in recognizing the substrate DNA sequence, in recognizing the other substrates, and in carrying out the chemical reactions of methylation or cleavage.