Open AccessCrystallization and preliminary X‐ray diffraction analysis on the homing endonuclease I‐Dmo‐I in complex with its target DNA
Author(s) -
Redondo Pilar,
Prieto Jesús,
Ramos Elena,
Blanco Francisco J.,
Montoya Guillermo
Publication year - 2007
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309107049706
Subject(s) - endonuclease , homing endonuclease , crystallography , dna , monoclinic crystal system , crystallization , escherichia coli , chemistry , x ray crystallography , diffraction , gene , physics , crystal structure , biochemistry , optics , organic chemistry
Homing endonucleases are highly specific DNA‐cleaving enzymes that recognize long stretches of base pairs. The availability of these enzymes has opened novel perspectives for genome engineering in a wide range of fields, including gene therapy, by taking advantage of the homologous gene‐targeting enhancement induced by a double‐strand break. I‐Dmo‐I is a well characterized homing endonuclease from the archaeon Desulfurococcus mobilis . The enzyme was cloned and overexpressed in Escherichia coli . Crystallization experiments of I‐Dmo‐I in complex with its DNA target in the presence of Ca 2+ and Mg 2+ yielded crystals that were suitable for X‐ray diffraction analysis. The crystals belonged to the monoclinic space group P 2 1 , with unit‐cell parameters a = 106.75, b = 70.18, c = 106.85 Å, α = γ = 90, β = 119.93°. The self‐rotation function and the Matthews coefficient suggested the presence of three protein–DNA complexes per asymmetric unit. The crystals diffracted to a resolution limit of 2.6 Å using synchrotron radiation at the Swiss Light Source (SLS) and the European Synchrotron Radiation Facility (ESRF).