Genes for Restriction Endonucleases and Modification Methylases

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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.