The domain organization of NaeI endonuclease:: Separation of binding and catalysis

NaeI is a remarkable type II restriction en donuclease. It must bind two recognition sequences to cleave DNA, forms a covalent protein-DNA intermediate, and is only 1 aa change away from topoisomerase and recombinase activity, The latter activities apparently derive from reactivation of a cryptic DNA ligase active site, Here, we demonstrate that NaeI has two protease-resistant domains, involving approximately the N-terminal and C-terminal halves of the protein, linked by a protease-accessible region of 30 aa, The domains were purified by cloning, The C-terminal domain was shown by gel mobility-shift assay to have approximately 8-fold lower DNA-binding ability than intact NaeI, Analytical ultracentrifugation showed this domain to be a monomer in solution, The N-terminal domain, which contains the catalytic region defined by random mutagenesis, did not bind DNA and was a mixture of different-sized complexes in solution implying that it mediates self-association. DNA greatly inhibited proteolysis of the linker region, The results identify the DNA-binding domain, imply that DNA cleavage and recognition are independent and separable, and lead us to speculate about a cleft-like structure for NaeI.