Biochemical characterization of I-CmoeI reveals that this H-N-H homing endonuclease shares functional similarities with H-N-H colicins

Endonuclease assays of the H-N-H proteins encoded by two group I introns in the Chlamydomonas moewusii chloroplast psbA gene revealed that the CmpsbA.1 intron specifies a site-specific DNA endonuclease, designated I-Cmoel. Like most previously reported intron-encoded endonucleases, I-Cmoel generates a; double-strand break near the insertion site of, its encoding intron, leaving 3' extensions of 4 nt. This enzyme was purified from Escherichia coli as a fusion protein with a His tag at its N-terminus. The recombinant protein (rl-Cmoel) requires a divalent alkaline earth cation for DNA cleavage (Mg(2+) > Ca(2+) > Sr(2+) > Ba(2+)). It,also requires a metal cofactor for DNA binding,la property shared with H-N-H colicins but not with-the homing endonucleases characterized to date. rl-model binds its recognition sequence as a monomer, as revealed by gel retardation assays, K(m) and k(cat) values of 100 +/- 40 pM and 0.26 +/- 0.04 min(-1), respectively, were determined, Replacement of the first histidine of the H-N-H motif by an alanine residue abolishes both rl-Cmoel activity and binding to its substrate. We propose that this conserved histidine residue plays a role in binding the metal cofactor and that such binding induces a structural modification of the enzyme which is required for DNA recognition.