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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 generat...

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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
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Lucas, P
Otis, C
Lemieux, C
Turmel, M
description 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.
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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
title Biochemical characterization of I-CmoeI reveals that this H-N-H homing endonuclease shares functional similarities with H-N-H colicins
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