We will develop a novel method for site-specific cleavage of double-stranded DNA for in vitro and in vivo applications. This method will create a new class of DNA restriction enzymes that can be designed by the researcher to specifically recognize a 20 base pair or longer unique sequences within a genome or other large stretch of DNA. These new enzymes will become powerful tools for in vitro and in vivo cleavage of whole genome DNAs. They will allow precise control over processes such as """"""""knock-out"""""""" gene experiments, facilitating homologous recombination in eukaryotic cells, and enabling re-engineering and/or cloning of large DNAs in vitro for various purposes, including functional genomics work. The novel method involves two sets of elements. The first are biotinylated targeting oligonucleotides (TONs) that are homologous to the sequences on both strands of the DNA to be cleaved. The TONs will be incubated with the target DNA in the presence of recA protein, which facilitates the formation of """"""""D -loop"""""""" complexes. In these D-loops the TONs are hybridized to exact recognition sequences within the double-stranded DNA, displacing the complementary strand of the DNA only in that region. The second element in this scheme is the catalytic subunit of Fok I, a type IIS restriction enzyme, which has been shown to cleave DNA when bound (3,4,5). We will genetically engineer the Fok I N-terminal (Fn) catalytic subunit so that it will be fused to an E. coli biotinylation peptide at one of its ends. This sequence causes E. coli to bioltinylate the expressed protein to which it is fused. This fused Fn protein will cleave DNA only when bound in a complex with streptavidin that is in turn bound to the hybridized biotinylated TONs described above. The combination of the recognition and cleavage elements of this scheme will create """"""""designer"""""""" restriction enzymes that will allow cleavage of DNA at precisely selected sites. Prior to Phase I, we successfully cloned and expressed the engineered Fokl (Fn) protein and showed that it is enzymatically active. The recA mediated TON insertion reaction was demonstrated with a plasmid template. In Phase I, we will improved the expression and purification of the biotinylated Fn enzyme, and measure the recA-mediated binding of the TONs to large segments of DNA. The two components will be tested to determine if they specifically cleave large DNAs. The resulting products, as kits and services, are applicable to 45,000 molecular biology labs worldwide. ? ? ?
