FURTHER CHARACTERIZATION OF ESCHERICHIA-COLI ENDONUCLEASE-V - MECHANISM OF RECOGNITION FOR DEOXYINOSINE, DEOXYURIDINE, AND BASE MISMATCHES IN DNA

Endonuclease V from Escherichia coli has a wide substrate spectrum. In addition to deoxyinosine-containing DNA, the enzyme cleaves DNA conta ining urea residues, AP sites, base mismatches, insertion/deletion mis matches, flaps, and pseudo-Y structures. The gene coding for the enzym e was identified to be orf 225 or nfi (endonuclease five). Using enzym e purified from an overproducing strain, the deoxyinosine-and mismatch -specific activities of endonuclease V was found to have different div alent metal requirements. The affinity of the enzyme is greater than 2 0-fold higher for DNA containing deoxyinosine than deoxynebularine or base mismatches. Under optimal cleavage conditions, endonuclease V for ms two stable complexes with DNA containing deoxyinosine, but not with DNA containing base mismatches or deoxynebularine, suggesting that th e 6-keto group of hypoxanthine in DNA is critical for stable interacti ons with the protein. The enzyme recognizes deoxyuridine in DNA but ex hibits a much lower affinity to DNA containing deoxyuridine compared w ith DNA containing deoxyinosine. Interestingly, deoxyuridine-specific endonuclease activity of endonuclease V has a divalent metal requireme nt similar to the mismatch activity, A model for the mechanism of subs trate recognition is proposed to explain these different activities.