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. 1997 Jul 1;25(13):2610–2619. doi: 10.1093/nar/25.13.2610

Evolutionarily conserved and functionally important residues in the I-CeuI homing endonuclease.

M Turmel 1, C Otis 1, V Côté 1, C Lemieux 1
PMCID: PMC146779  PMID: 9185572

Abstract

Two approaches were used to discern critical amino acid residues for the function of the I- Ceu I homing endonuclease: sequence comparison of subfamilies of homologous proteins and genetic selection. The first approach revealed residues potentially involved in catalysis and DNA recognition. Because I- Ceu I is lethal in Escherichia coli , enzyme variants not perturbing cell viability were readily selected from an expression library. A collection of 49 variants with single amino acid substitutions at 37 positions was assembled. Most of these positions are clustered within or around the LAGLI-DADG dodecapeptide and the TQH sequence, two motifs found in all protein subfamilies examined. The Km and kcat values of the wild-type and nine variant enzymes synthesized in vitro were determined. Three variants, including one showing a substitution of the glutamine residue in the TQH motif, revealed no detectable endonuclease activity; five others showed reduced activity compared to the wild-type enzyme; whereas the remaining variant cleaved the top strand about three times more efficiently than the wild-type. Our results not only confirm recent reports indicating that amino acids in the LAGLI-DADG dodecapeptide are functionally critical, but they also suggest that some residues outside this motif directly participate in catalysis.

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Selected References

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