PMID: 9185572Jul 1, 1997Paper

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

Nucleic Acids Research
Monique TurmelClaude Lemieux

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, ...Continue Reading

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Citations

Oct 17, 2002·Journal of Molecular Biology·Marilena Fitzsimons HallIra Schildkraut
Feb 13, 2001·Nucleic Acids Research·P LucasC Lemieux
Aug 31, 2002·Nucleic Acids Research·Lenny M SeligmanAdeline L Veillet
Apr 8, 2004·Nucleic Acids Research·Peik Haugen, Debashish Bhattacharya
Mar 17, 2004·Genetics·Anya BakhratDina Raveh
Aug 30, 2003·Proceedings of the National Academy of Sciences of the United States of America·Camilla L Nesbø, W Ford Doolittle
Nov 17, 2009·Cellular and Molecular Life Sciences : CMLS·Maria J MarcaidaGuillermo Montoya
Dec 5, 2006·Journal of Virological Methods·Olga A LihoradovaJeffrey M Slack
May 12, 2005·The FEBS Journal·Sayuri KurokawaTakeshi Ohama
Nov 11, 1998·Molecular Cell·M S JuricaB L Stoddard
Jul 27, 2002·Annual Review of Microbiology·J Peter GogartenElena Hilario
Apr 3, 1999·The Journal of Biological Chemistry·V PingoudA Pingoud
Nov 7, 1998·The Journal of Biological Chemistry·F S GimbleF A Quiocho
Jun 10, 2017·Current Stem Cell Reports·Juliette M K M Delhove, Waseem Qasim
Aug 10, 1999·Journal of Bacteriology·K D EverettM G Friedman

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