Functional characterization of isoschizomeric His-Cys box homing endonucleases from Naegleria

Several species within the amoeboflagellate genus Naegleria harbor an optio nal ORF containing group I introns in their nuclear small subunit ribosomal DNA. The different ORFs encode homing endonucleases with 65 to 95% identit y at the amino-acid level. I-NjaI, I-NanI and I-NitI, from introns in Naegl eria jamiesoni, N. andersoni and N. italica, respectively, were analyzed in more detail and found to be isoschizomeric endonucleases that recognize an d cleave an approximal 19-bp partially symmetrical sequence, creating a pen tanucleotide 3' overhang upon cleavage. The optimal conditions for cleavage activity with respect to temperature, pH, salt and divalent metal ions wer e investigated. The optimal cleavage temperature for all three endonuclease s was found to be 37 degreesC and the activity was dependent on the concent ration of NaCl with an optimum at 200 mM. Divalent metal ions, primarily Mg 2+, essential for Naegleria endonuclease activity. Whereas both Mn2+ and Ca 2+ could substitute for Mg2+, but with a slower cleavage rate, Zn2+ was una ble to support cleavage. Interestingly, the pH dependence of DNA cleavage w as found to vary significantly between the I-NitI and I-NjaI/I-NanI endonuc leases with optimal pH values at 6.5 and 9, respectively. Site-directed mut agenesis of conserved I-NjaI residues strongly supports the hypothesis that Naegleria homing endonucleases share a similar zinc-binding structure and active site with the His-Cys box homing endonuclease I-PpoI.