How are Restriction Systems Controlled, and How Do They Recognize DNA Sequences?

Blumenthal限制性内切酶（REases）是在特定核苷酸序列上切割双链DNA的酶。产生酶的细菌必须保护自己的DNA，这通常是通过产生甲基转移酶（MTase）来完成的，甲基转移酶将一个甲基从s -腺苷- l-蛋氨酸转移到DNA上：酶不会切割已经适当甲基化的DNA序列。有两个问题很突出。首先，ase及其同源mtase的基因通常位于质粒等可移动的遗传元件上。当这样的质粒被转移到新的宿主细胞中时，如何控制基因和酶以确保新宿主的DNA在酶活性出现之前被甲基化？对于任何release / MTase对，这个问题都没有得到完全的回答。这个项目的一个主要目标是为pvii release / MTase对回答这个问题。该实验室已经发现了影响PvuII的三个特征：PvuII酶的mRNA中的预测发夹可能调节翻译的起始；一种明显的转录激活因子是PvuII REase基因表达所必需的，一种小蛋白似乎可以阻止PvuII REase的两个相同亚基相互结合。关于ase和mtase的第二个主要问题是它们如何在所有存在的DNA中识别它们的底物核苷酸序列。ase和mtase不仅要与特定的DNA序列结合，还必须使DNA分子的相关部分与活性位点接触。这是否对如何识别DNA序列施加了特殊的限制？一种方法是比较识别相同序列（或密切相关序列）的几种蛋白质。本项目试图通过以下蛋白质来表征DNA序列识别，“^”表示切割，5mC和N4mC表示在指示位置的胞嘧啶甲基化：PvuII REase CAG^CTG PvuII MTase CAGN4mCTG AluI Ease AG^CT AluI MTase AG5mCT这些蛋白质将与其他研究的基本区域螺旋-环-螺旋转录因子进行比较，这些转录因子识别序列CAGCTG但不催化任何反应。某些细菌产生特殊的DNA修饰酶，即限制性内切酶（REase），它能破坏其他细菌或病毒的DNA。对生物体DNA的保护是由另一种类型的DNA修饰酶——甲基转移酶来完成的，甲基转移酶会改变DNA的化学性质，这样酶就不会破坏产生酶的细菌的DNA。在生物研究中，利用激光在精确的位置切割DNA，就像用显微激光解剖基因一样。该项目将深入了解这些修饰酶的调控、结构和功能，为最终设计新的分子生物学工具提供信息，并能够设计出操纵细菌遗传转移的机制。＊＊＊

9631137 Blumenthal Restriction endonucleases (REases) are enzymes that cleave both strands of double-stranded DNA at specific nucleotide sequences. Bacteria that produce REases must protect their own DNA, and this is generally done by producing a methyltransferase (MTase) that transfers a methyl group from S-adenosyl-L-methionine to the DNA: the REases do not cleave DNA sequences that have been properly methylated. Two questions stand out. First, the genes for REases and their cognate MTases are often located on mobile genetic elements such as plasmids. When such a plasmid is transferred into a new host cell, how are the genes and enzymes controlled to ensure that the new host's DNA is methylated before REase activity appears? This question has not been fully answered for any REase / MTase pair. One major goal of this project is to answer this question for the PvuII REase / MTase pair. The proposing laboratory has discovered three features affecting PvuII a predicted hairpin in the mRNA for the PvuII REase which may modulate initiation of translation; an apparent transcriptional activator that is required for expression of the PvuII REase gene and a small protein that appears to prevent the two identical subunits of PvuII REase from associating with one another. The second major question regarding REases and MTases is how they recognize their substrate nucleotide sequence amongst all of the other DNA that is present. REases and MTases must not only bind to a specific DNA sequence, but must then bring the relevant portions of the DNA molecule into contact with an active site or sites. Does this impose particular constraints on how the DNA sequence is recognized? One way to approach this is to compare several proteins that recognize the same sequence (or closely related sequences). This project seeks to characterize the DNA sequence recognition by the following proteins "^" indicates cleavage, 5mC and N4mC indicate methylation of the cytosine at the indicated position: PvuII REase CAG^CTG PvuII MTase CAGN4mCTG AluI Ease AG^CT AluI MTase AG5mCT These proteins will be compared to those basic region helix-loop-helix transcription factors, studied by others, which recognize the sequence CAGCTG but do not catalyze any reaction. %%% Certain bacteria produce specialized DNA modifying enzymes, restriction endonucleases (REase), that destroy DNA from other bacteria or viruses. Protection of the organism's DNA is done by a different type of DNA modifying enzymes, methytransferases, that change the chemical nature of the DNA so that the REase does not destroy the DNA of the bacteria that produces the REase. REases are utilized in biological research to cut DNA at precise locations like microscopic lasers to dissect genes. This project will provide insight into the regulation, structure, and function of these modifying enzymes providing information for the eventual design of new molecular biological tools, and the ability to devise mechanisms to manipulate genetic transfer in bacteria. ***