Location of putative binding and catalytic sites of NaeI by random mutagenesis.

Endonuclease NaeI is a prototype for an unusual group of type II restriction endonucleases that must bind two DNA recognition sequences to cleave DNA. The naeIR gene, expressed from a Ptac promotor construct, was toxic to Escherichia coli in the absence of NaeI-sequence specific methylases. The naeIR gene was mutagenized with N-methyl-N'-nitrosoguanidine; four classes of NaeI variants were isolated in the absence of protecting methylase activity. Class I variants (T60I, E70K) lacked detectable cleavage activity, but displayed good sequence-specific DNA binding. Class II variants (D95N, G141D) displayed 1-5% of the wild-type cleavage activity and normal DNA binding. Class III variants (G131E, G131R, G155D, G245E) displayed significantly attenuated cleavage and binding activities. Class IV variants (G197D, G214R/A219T, G236S, L241P, G245E, G245R, G250E, G270E) lacked both cleavage and binding activities. These results imply two amino acids (Thr-60, Glu-70) essential for catalysis. In addition, two domains are indicated in NaeI: one (Thr-60 to Gly-155) mediates substrate binding and catalysis, the other (Gly-197 to Gly-270) may mediate binding of the activating DNA sequence. Our results are compared with the active site residues of EcoRI, EcoRV, and BamHI.