The domain organization of Nae I endonuclease: Separation of binding and catalysis

• Published in: Proceedings of the National Academy of Sciences - PNAS 1998-03, Vol.95 (7), p.3531-3536
• Main Authors: Colandene, James D., Topal, Michael D.
• Format: Article
• Language: English
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.95.7.3531

Nae I is a remarkable type II restriction endonuclease. 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 Nae I 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 Nae I. 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 Nae I.