Genes for Restriction Endonucleases and Modification Methylases

这个项目的长期目标是了解限制酶和修饰酶如何在识别DNA序列时实现其精致的特异性。为了实现这一目标，将研究一种在结构和功能上都相关的酶。大量限制性内切酶和修饰酶的序列比较表明，限制性内切酶之间在序列水平上没有明显的相似之处。在产生N6-甲基腺嘌呤的甲基酶之间有一些有限的相似性，但在产生C5-甲基胞嘧啶的甲基酶之间有很大的相似性。其中三种胞嘧啶甲基酶将被非常详细地研究。它们是MspI甲基酶(CCGG-MeCCGG)、HpaII甲基酶(CCGG-CMeCGG)和Hhai甲基酶(GCGC-GMeCGC)。已经制备了MspI甲基酶的高表达克隆，并且该甲基酶已经纯化到接近均一的水平。这种蛋白质的结晶，无论是单独的结晶，还是与DNA的络合结晶，都将被尝试。HpaII甲基酶基因的序列已经完成，与MspI甲基酶基因和Hhai甲基酶基因都有显著的相似性。未来三年的主要目标是详细定义一个或所有这些甲基酶基因的识别结构域。这将通过尝试在三种甲基酶之间进行一系列结构域交换实验来实现。这些结构域交换将包括在选定的限制性内切酶位点的精确交换和定义较不明确的交换，随后进行遗传筛选以保护重组甲基酶。如果能够分离出能够与DNA结合的功能结构域，将制备并测试蛋白质的截断版本。

The long term goal of this project is to understand how restriction and modification enzymes achieve their exquisite specificity in recognizing DNA sequences. To achieve this goal set a enzymes that are related both structurally and functionally will be studied. Comparison of the sequences for a large number of restriction and modification enzymes shows that among restriction enzymes there are no apparent similarities at the sequence level. Among methylases that produce N6-methyladenine there is some limited similarity, but among methylases that produce C5-methylcytosine there is a great deal of similarity. Three of these cytosine methylases will be studied in great detail. These are the MspI methylase (CCGG - MeCCGG), the HpaII Methylase (CCGG - CMeCGG) and the HhaI methylase (GCGC - GMeCGC). An overexpressing clone of the MspI methylase has been prepared and the methylase already purified to near-homogeneity. The crystallization of this protein both alone and complexed with DNA will be attemped. The sequence of the HpaII methylase gene has already been completed and it shows striking similarities both to the MspI methylase gene and the HhaI methylase gene. A major goal of the next three year period is to define in detail the recognition domains of one or all of these methylase genes. This will be accomplished by attempting a series of domain swap experiments between the three methylases. These domain swaps will involv both exact swaps at selected restriction enzyme sites and less well- defined swaps followed by genetic screening to dtect recombinant methylases. Truncated versions of the proteins will be prepared and tested if functional domains can be isolated that are able to bind DNA.