We propose to develop a novel system for directed nucleic acid cleavage. This system is based upon our discovery that the 5' nucleases of several thermostable DNA polymerases can be directed to cleave as site-specific endonucleases. This system will be thermostable, specific, efficient and easily automatable, and will find broad application in the fields of nucleic acids-based research and diagnostics. In Phase I we will seek to overcome the most substantial limitation of this technology, its inefficiency at cleaving long 5' arms. We will examine the effects of reaction constituents and conditions on the efficiency of cleavage of a well-characterized substrate, performing a comprehensive examination of the effects of different salts, divalent cations, denaturants, buffer pH values, helicases and temperatures. We will also use mutagenesis to alter the protein and influence its interaction with the substrate structure. During Phase II we will optimize the required substrate complex formations and nuclease interactions to develop this technology into a generic, directed nucleic acid cleavage system for both single and double stranded nucleic acids.