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

该项目的长期目标是了解蛋白质如何识别特定的DNA序列，以及识别机制如何变化以适应蛋白质的功能需求。为此，突变体表征和蛋白质生物化学的结合将用于研究三种功能不同的dna结合蛋白，它们都来自Pvu II限制性修饰系统。这些蛋白质中的第一个是Pvu II DNA甲基转移酶，它结合s -腺苷甲硫氨酸和DNA双链序列CAGCTG，并甲基化内部胞嘧啶的氨基。第二种蛋白质是Pvu II限制性内切酶，它结合相同的DNA序列和mg++，并催化中心两个碱基之间的磷酸二酯裂解。第三种蛋白是Pvu II C蛋白，它似乎对DNA具有更退化的识别特异性，并激活核酸内切酶基因的转录。当前的目标是确定在识别底物DNA序列和其他底物中起关键作用的结构域和氨基酸。了解蛋白质对DNA的序列特异性识别，特别是当催化中心必须靠近DNA时，是对基因表达的复杂理解和生物技术的进一步发展的重要目标。这个项目的两个长期目标是了解蛋白质如何识别特定的DNA碱基序列，尽管细胞中有大量的非底物DNA，以及当催化中心必须靠近DNA进行反应时，这些序列识别机制如何变化以适应蛋白质的其他功能需求。了解蛋白质对DNA的序列特异性识别是全面了解基因表达调控和进一步发展生物技术的重要目标。本研究将利用诱变和蛋白质生物化学相结合的方法研究Pvu II限制性修饰系统中三种功能各异的dna结合蛋白。这些蛋白质中的第一个是Pvu II DNA甲基转移酶，它结合s -腺苷甲硫氨酸和DNA双链序列CAGCTG，并甲基化内部胞嘧啶的氨基。第二种蛋白质是Pvu II限制性内切酶，它将相同的DNA序列与Mg++结合，并催化中心两个碱基之间的磷酸二酯裂解。第三种蛋白是Pvu II C蛋白，它似乎结合Pvu II DNA并激活核酸内切酶基因的转录。当前的目标是确定这三种蛋白质的结构域和氨基酸，它们在识别底物DNA序列、识别其他底物以及进行甲基化或切割的化学反应中发挥关键作用。

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.