Generation and analysis of mesophilic variants of the thermostable archaeal I-DmoI homing endonuclease.
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 degrees 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.
Prieto
Jesús
J
Structural Biology and Biocomputing Programme, NMR Group and Macromolecular Crystallography Group, Spanish National Cancer Center, c/Melchor Fernández Almagro 3, 28029-Madrid, Spain.
Epinat
Jean-Charles
JC
Redondo
Pilar
P
Ramos
Elena
E
Padró
Daniel
D
Cédrone
Frédéric
F
Montoya
Guillermo
G
Pâques
Frédéric
F
Blanco
Francisco J
FJ
D001483:Base Sequence; D017931:DNA Primers; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000378:metabolism*; D019620:Desulfurococcaceae / Q000201:enzymology; D004795:Enzyme Stability; D008958:Models, Molecular; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014461:Ultracentrifugation
D017931:DNA Primers; D004262:DNA Restriction Enzymes