NaeI, a DNA endonuclease from an aerobic actinomycete, is a prototype for a new type of sequence-specific endonuclease that offers opportunities for new information about protein-DNA interactions and possibilities of new technologies. NaeI must bind an enhancer DNA element to cleave its recognition site (Fig.1): Enhancer and substrate elements are distinguished by the sequences immediately surrounding a core six-base recognition site that is required for binding. Enhancer induces assembly of an active cleavasome that involves conformational changes to the protein to activate catalysis. The characteristics of the NaeI-type enzymes and possible homology between one member, EcoRII, and the Int-family of recombinases indicates that this type of enzyme may be an evolutionary link between the restriction enzymes and the recombinases. The long-term objectives of this application are to use a protein engineering approach to alter the DNA specificity of NaeI endonuclease to recognize pathologic sequences in human DNA. Near term we will use molecular biology to map and characterize the functional domains of NaeI.
Specific Aims i nclude: 1) Mutate and select for different classes of NaeI mutants. 2) Select for mutations in the region required for NaeI dimerization. 3) Map the mutations to specific regions of the gene and determine the mutagenic changes to DNA sequence. 4) Site-directed mutagenesis of recognized functional domains. 5) Biochemical Characterization of the Mutations. 6) Change the specificity of NaeI-DNA recognition.
