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-CMOE:I. Like most previously reported intron-encoded endonucleases, I-CMOE:I 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-CMOE:I) 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, a property shared with H-N-H colicins but not with the homing endonucleases characterized to date. rI-CMOE:I 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 rI-CMOE:I 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.
Drouin
M
M
Centre de Recherche sur la Fonction, la Structure et l'Ingénierie des Protéines, Université Laval, Québec G1K 7P4, Canada.
Lucas
P
P
Otis
C
C
Lemieux
C
C
Turmel
M
M
D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000494:pharmacology; D002696:Chlamydomonas / Q000201:enzymology / Q000235:genetics; D003087:Colicins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000187:drug effects / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005785:Gene Conversion; D006639:Histidine / Q000235:genetics; D007438:Introns; D007700:Kinetics; D009154:Mutation; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics; D045332:Photosystem II Protein Complex; D011485:Protein Binding; D013379:Substrate Specificity
D020418:Algal Proteins; D002413:Cations, Divalent; D003087:Colicins; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D006639:Histidine; D004247:DNA; D004706:Endodeoxyribonucleases; D004262:DNA Restriction Enzymes