THE DOMAIN ORGANIZATION OF NAEI ENDONUCLEASE - SEPARATION OF BINDING AND CATALYSIS

NaeI is a remarkable type II restriction en donuclease. It must bind t wo recognition sequences to cleave DNA, forms a covalent protein-DNA i ntermediate, and is only 1 aa change away from topoisomerase and recom binase activity, The latter activities apparently derive from reactiva tion of a cryptic DNA ligase active site, Here, we demonstrate that Na eI has two protease-resistant domains, involving approximately the N-t erminal and C-terminal halves of the protein, linked by a protease-acc essible region of 30 aa, The domains were purified by cloning, The C-t erminal domain was shown by gel mobility-shift assay to have approxima tely 8-fold lower DNA-binding ability than intact NaeI, Analytical ult racentrifugation showed this domain to be a monomer in solution, The N -terminal domain, which contains the catalytic region defined by rando m mutagenesis, did not bind DNA and was a mixture of different-sized c omplexes in solution implying that it mediates self-association. DNA g reatly inhibited proteolysis of the linker region, The results identif y 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.