Crystallization and preliminary X-ray diffraction analysis on the homing endonuclease I-Dmo-I in complex with its target DNA.

Abstract

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 P2(1), with unit-cell parameters a = 106.75, b = 70.18, c = 106.85 A, alpha = gamma = 90, beta = 119.93 degrees . 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 A using synchrotron radiation at the Swiss Light Source (SLS) and the European Synchrotron Radiation Facility (ESRF).