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

• Published in: Nucleic acids research 2000-11, Vol.28 (22), p.4566-4572
• Main Authors: Drouin, M, Lucas, P, Otis, C, Lemieux, C, Turmel, M
• Format: Article
• Language: English
• Subjects: Algal Proteins - genetics; Algal Proteins - metabolism; Amino Acid Substitution; Animals; barium; Base Sequence; Binding Sites; Cations, Divalent - pharmacology; Chlamydomonas - enzymology; Chlamydomonas - genetics; Chlamydomonas moewusii; Colicins - chemistry; Colicins - genetics; Colicins - metabolism; deoxyribonuclease I-CmoeI; DNA - genetics; DNA - metabolism; DNA Restriction Enzymes - drug effects; DNA Restriction Enzymes - metabolism; Endodeoxyribonucleases - chemistry; Endodeoxyribonucleases - genetics; Endodeoxyribonucleases - metabolism; Escherichia coli; Gene Conversion; Histidine - genetics; homing endonuclease; Introns; Kinetics; Mutation; Photosynthetic Reaction Center Complex Proteins - genetics; Photosystem II Protein Complex; Protein Binding; strontium; Substrate Specificity
• ISSN: 1362-4962; 0305-1048; 1362-4962
• DOI: 10.1093/nar/28.22.4566

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.