Restriction enzymes provide excellent models for the study of DNA binding enzymes due to their abundance and diversity. EcoRII endonuclease and methylase are a restriction enzyme pair that recognizes the base sequence CC(A or T)GG. There are over forty other restriction systems known that recognize this same sequence. We propose to investigate how the EcoRII methylase recognizes its cannonical sequence and how it transfers the methyl group to this sequence. We plan to use both biochemical and genetic means for this purpose and develop methods that will be useful in studying all cytosine methylases. EcoRII genes have been cloned and sequenced, the proteins purified to homogeneity and a considerable body of data exists on the reaction mechanism of the methylase. This makes EcoRII methylase an attractive choice for study among methylases. We will isolate mutant methylases that are blocked in a specific step in methyl-transfer and mutants that recognize an altered base sequence, and determine the sites of mutations. We will also map the amino acid residues involved in the binding of the co-factor S-adenosyl methionoine and those involved in a reaction step chemically. Different DNA cytosine methylases appear to have similar overall structure, regardless of the sequence they recognize. We plan to test the hypothesis that the regions of sequence conservation between cytosine methylases are involved in the common methyl-transfer reaction, while the sequences unique to each enzyme are involved in base sequence recognition. If this hypothesis is confirmed, insights gained during the study of EcoRII are likely to be of broader use and a way may be opened up to make cytosine methylases with novel sequence specificities.
