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

Content Provider	PubMed Central
Author	Drouin, Mathieu Lucas, Patrick Otis, Christian Lemieux, Claude Turmel, Monique
Copyright Year	2000
Abstract	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. 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-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.
Starting Page	4566
File Format	PDF
ISSN	13624962
e-ISSN	13624962
Journal	Nucleic Acids Research
Issue Number	22
Volume Number	28
Language	English
Publisher	Oxford University Press
Publisher Date	2000-11-15
Access Restriction	Open
Rights Holder	Oxford University Press
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Genetics

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