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Published online 9 June 2004

Nucleic Acids Research, 2004, Vol. 32, No. 10 3156-3168
© 2004 Oxford University Press

Analysis of the LAGLIDADG interface of the monomeric homing endonuclease I-DmoI

George H. Silva and Marlene Belfort*

Wadsworth Center, New York State Department of Health and State University of New York at Albany, Albany, NY 12201-2002, USA

*To whom correspondence should be addressed. Tel: +1 518 473 3345; Fax: +1 518 474 3181; Email: belfort{at}wadsworth.org
Present address:
George H. Silva, Institut fuer Biochemie, Justus-Liebig-Universitaet, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany

Received February 27, 2004; Revised and Accepted April 30, 2004

The general structural fold of the LAGLIDADG endonuclease family consists of two similar {alpha}/ß domains ({alpha}ßß{alpha}ßß{alpha}) that assemble either as homodimers or monomers with the domains related by pseudo-two-fold symmetry. At the center of this symmetry is the closely packed LAGLIDADG two-helix bundle that forms the main inter- or intra-molecular contact region between the domains of single- or double-motif proteins, respectively. In this work, we further examine the role of the LAGLIDADG residues involved in the helix–helix interaction. The interchangeability of the LAGLIDADG helix interaction was explored by grafting interfacial residues from the homodimeric I-CreI into the corresponding positions in the monomeric I-DmoI. The resulting LAGLIDADG exchange mutant is partially active, preferring to nick dsDNA rather than making the customary double-strand break. A series of partial revertants within the mutated LAGLIDADG region are shown to restore cleavage activity to varying degrees resulting in one I-DmoI mutant that is more active than wild-type I-DmoI. The phenotype of some of these mutants was reconciled on the basis of similarity to the GxxxG helix interaction found in transmembrane proteins. Additionally, a split variant of I-DmoI was created, demonstrating that the LAGLIDADG helices of I-DmoI are capable of forming and maintaining the protein–protein interface in trans to create an active heterodimer.


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