Our long term objective is to understand sequence specific recognition of DNA by proteins using FokI restriction-modification enzymes as a model system. FokI is a Type IIS restriction enzyme which recognizes the nonpalindromic pentanculeotide duplex, d- GGATG:d-CATCC and cleaves about 9/13 bp away from the recognition sequence. This implies the presence of two separate protein domains in the enzyme: one for sequence specific recognition and the other for the endonuclease activity. Once the DNA binding domain anchors at the appropriate site, a signal is transmitted to the endonuclease, probably through allosteric interactions and the cleavage occurs. The FokI restriction-modification genes from Flavobacterium Okeanokoites (IFO 12536) will be cloned into pBR322. A restriction fragment containing the FokI restriction gene will be inserted at the BamHI cloning site of plamid pDR540. This plasmid has been modified by insertion of the lac iq gene into the EcoRI site. For overproduction, the recombinants will be transfected into RB79iq cells carrying another compatible plasmid containing the FokI methylase gene that is expressed constitutively. After induction with IPTG, the FokI restriction enzyme will be purified to homogeneity. We will also delete portions of the gene to isolate the smallest protein domain that will bind the DNA in a sequence specific manner. Once we have sufficient amount of these proteins, we will initiate NMR studies to obtain information about the structural details and chemical dynamics of this system.
