Open AccessFunctional characterization of isoschizomeric His‐Cys box homing endonucleases from Naegleria
Author(s) -
Elde Morten,
Willassen Nils P.,
Johansen Steinar
Publication year - 2000
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2000.01862.x
Subject(s) - homing endonuclease , endonuclease , cleavage (geology) , naegleria , biology , divalent metal , divalent , dna , enzyme , biochemistry , chemistry , stereochemistry , metal , genetics , paleontology , protozoa , organic chemistry , fracture (geology)
Several species within the amoeboflagellate genus Naegleria harbor an optional ORF containing group I introns in their nuclear small subunit ribosomal DNA. The different ORFs encode homing endonucleases with 65 to 95% identity at the amino‐acid level. I‐ Nja I, I‐ Nan I and I‐ Nit I, from introns in Naegleria jamiesoni , N. andersoni and N. italica , respectively, were analyzed in more detail and found to be isoschizomeric endonucleases that recognize and cleave an approximal 19‐bp partially symmetrical sequence, creating a pentanucleotide 3′ overhang upon cleavage. The optimal conditions for cleavage activity with respect to temperature, pH, salt and divalent metal ions were investigated. The optimal cleavage temperature for all three endonucleases was found to be 37 °C and the activity was dependent on the concentration of NaCl with an optimum at 200 m m . Divalent metal ions, primarily Mg 2+ , are essential for Naegleria endonuclease activity. Whereas both Mn 2+ and Ca 2+ could substitute for Mg 2+ , but with a slower cleavage rate, Zn 2+ was unable to support cleavage. Interestingly, the pH dependence of DNA cleavage was found to vary significantly between the I‐ Nit I and I‐ Nja I/I‐ Nan I endonucleases with optimal pH values at 6.5 and 9, respectively. Site‐directed mutagenesis of conserved I‐ Nja I 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‐ Ppo I.