The project encompasses the investigation of the molecular basis of sequence specific recognition by DNA binding proteins and, in particular, the mechanism by which type II endonucleases achieve their high degree of binding and catalytic specificity. SmaI, XmaI and XcyI each recognize the hexanucleotide CCCGGG. SmaI cleaves between the internal CpG, while XmaI and XcyI are perfect isoschizomers and cleave between the external cytosines. Cytosine methylation within the recognition site inhibits the activity of the enzymes. It is the aim of this proposal to focus on the DNA binding properties of the endonucleases and to compare the mechanism by which each of the proteins interacts with the same DNA sequence. The project involves structural characterization of the endonucleases and analysis of the protein-DNA interaction in terms of the overall topology of the protein-DNA complexes, specific base and phosphate contacts and the potential for protein-induced bending and unwinding of the DNA. The effect of cytosine methylation on the binding properties of the endonucleases will also be investigated. The comparative study of the endonucleases will reveal whether there are any similarities in their mechanism of molecular recognition or whether each is characterized by a distinct mode of interaction with DNA. Furthermore, the enzymes also provide a good model system for analyzing the potential roles of cytosine methylation in protein-DNA interactions.
