HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 7, 4364-4374, February 15, 2008

This Article Full Text Full Text (PDF) All Versions of this Article: 283/7/4364    most recent M706323200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Prieto, J. Articles by Blanco, F. J. Search for Related Content PubMed PubMed Citation Articles by Prieto, J. Articles by Blanco, F. J. Social Bookmarking                      What's this?

Generation and Analysis of Mesophilic Variants of the Thermostable Archaeal I-DmoI Homing Endonuclease^*

Jesús Prieto¹, Jean-Charles Epinat¹, Pilar Redondo^¶, Elena Ramos, Daniel Padró², Frédéric Cédrone, Guillermo Montoya^¶, Frédéric Pâques³, and Francisco J. Blanco⁴

From the Structural Biology and Biocomputing Programme, NMR Group and ^¶Macromolecular Crystallography Group, Spanish National Cancer Center, c/Melchor Fernández Almagro 3, 28029-Madrid, Spain and Cellectis S.A., 102 Route de Noisy, 93235 Romainville, France

The hyperthermophilic archaeon Desulfurococcus mobilis I-DmoI protein belongs to the family of proteins known as homing endonucleases (HEs). HEs are highly specific DNA-cleaving enzymes that recognize long stretches of DNA and are powerful tools for genome engineering. Because of its monomeric nature, I-DmoI is an ideal scaffold for generating mutant enzymes with novel DNA specificities, similarly reported for homodimeric HEs, but providing single chain endonucleases instead of dimers. However, this would require the use of a mesophilic variant cleaving its substrate at temperatures of 37 °C and below. We have generated mesophilic mutants of I-DmoI, using a single round of directed evolution that relies on a functional assay in yeast. The effect of mutations identified in the novel proteins has been investigated. These mutations are located distant to the DNA-binding site and cause changes in the size and polarity of buried residues, suggesting that they act by destabilizing the protein. Two of the novel proteins have been produced and analyzed in vitro. Their overall structures are similar to that of the parent protein, but they are destabilized against thermal and chemical denaturation. The temperature-dependent activity profiles for the mutants shifted toward lower temperatures with respect to the wild-type activity profile. However, the most destabilized mutant was not the most active at low temperatures, suggesting that other effects, like local structural distortions and/or changes in the protein dynamics, also influence their activity. These mesophilic I-DmoI mutants form the basis for generating new variants with tailored DNA specificities.

Received for publication, July 31, 2007 , and in revised form, October 26, 2007.

^* This work was partly supported by the European Union Framework Programme for Research Contract LSHG-CT-2006-037226, MEGATOOLS. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Present address: CIC biomaGUNE, Paseo Miramón 182, 20009 San Sebastián, Spain.

³ To whom correspondence may be addressed. Tel.: 33141839914; Fax: 33141839903; E-mail: paques{at}cellectis.com.

⁴ To whom correspondence may be addressed: Structural Biology Unit, CIC bio-GUNE, Edificio 800, Parque Tecnológico de Bizkaia, 48160 Derio, Spain. Tel.: 34946572521; Fax: 34944061301; E-mail: fblanco{at}cicbiogune.es.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				R. Takeuchi, M. Certo, M. G. Caprara, A. M. Scharenberg, and B. L. Stoddard Optimization of in vivo activity of a bifunctional homing endonuclease and maturase reverses evolutionary degradation Nucleic Acids Res., February 1, 2009; 37(3): 877 - 890. [Abstract] [Full Text] [PDF]

				M. Sasaki, M. Uno, S. Akanuma, and A. Yamagishi Random mutagenesis improves the low-temperature activity of the tetrameric 3-isopropylmalate dehydrogenase from the hyperthermophile Sulfolobus tokodaii Protein Eng. Des. Sel., December 1, 2008; 21(12): 721 - 727. [Abstract] [Full Text] [PDF]

				M. J. Marcaida, J. Prieto, P. Redondo, A. D. Nadra, A. Alibes, L. Serrano, S. Grizot, P. Duchateau, F. Paques, F. J. Blanco, et al. Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering PNAS, November 4, 2008; 105(44): 16888 - 16893. [Abstract] [Full Text] [PDF]