Nucleic Acids Research, 2000, Vol. 28, No. 22 4566-4572
© 2000 Oxford University Press
Biochemical characterization of I-CmoeI reveals that this H-N-H homing endonuclease shares functional similarities with H-N-H colicins
Centre de Recherche sur la Fonction, la Structure et l’Ingénierie des Protéines, Université Laval, Québec G1K 7P4, Canada
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-CmoeI. Like most previously reported intron-encoded endonucleases, I-CmoeI 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 (rI-CmoeI) requires a divalent alkaline earth cation for DNA cleavage (Mg2+ > Ca2+ > Sr2+ > Ba2+). It also requires a metal cofactor for DNA binding, a property shared with H-N-H colicins but not with the homing endonucleases characterized to date. rI-CmoeI binds its recognition sequence as a monomer, as revealed by gel retardation assays. Km and kcat 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 rI-CmoeI 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.
* To whom correspondence should be addressed. Tel: +1 418 656 2131; Fax: +1 418 656 5036; Email: monique.turmel{at}rsvs.ulaval.ca
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