I-NjaI, a nuclear intron-encoded homing endonuclease from Naegleria, generates a pentanucleotide 3 ' cleavage-overhang within a 19 base-pair partially symmetric DNA recognition site

Different species of the amoebo-flagellate Naegleria harbor optional group I introns in the nuclear ribosomal DNA that contain open reading frames. In tron proteins from Naegleria jamiesoni, Naegleria andersoni, and Naegleria italica (named I-NjaI, I-NanI and I-NitI, respectively) were expressed in E scherichia coli and found to be isoschizomeric homing endonucleases that sp ecifically recognize and cleave intron-lacking homologous alleles of riboso mal DNA. The I-NjaI endonuclease was affinity purified, characterized in mo re detail, and found to generate five-nucleotide 3' staggered ends. at the intron insertion site which differs from the ends generated by all other kn own homing endonucleases. The recognition site was delimited and found to c over an approximate to 19 base-pair partially symmetric sequence spanning b oth the cleavage site and the intron insertion site. The palindromic featur e was supported by mutational analysis of the target DNA. All single-site s ubstitutions within the recognition sequence were cleaved by the purified I -NjaI endonuclease, but at different efficiencies. The center of symmetry a nd cleavage was found to be completely degenerate in specificity, which res embles that of the subclass IIW bacterial restriction enzymes.