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Crystallization and preliminary X‐ray diffraction analysis of the homing endonuclease I‐CvuI from Chlorella vulgaris in complex with its target DNA
Author(s) -
Redondo Pilar,
Merino Nekane,
Villate Maider,
Blanco Francisco J.,
Montoya Guillermo,
Molina Rafael
Publication year - 2014
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x1400065x
Subject(s) - homing endonuclease , endonuclease , dna , orthorhombic crystal system , crystallization , chlorella vulgaris , escherichia coli , crystallography , chemistry , biology , microbiology and biotechnology , gene , biochemistry , crystal structure , botany , organic chemistry , algae
Homing endonucleases are highly specific DNA‐cleaving enzymes that recognize long stretches of DNA. The engineering of these enzymes provides novel instruments for genome modification in a wide range of fields, including gene targeting, by inducing specific double‐strand breaks. I‐CvuI is a homing endonuclease from the green alga Chlorella vulgaris . This enzyme was purified after overexpression in Escherichia coli . Crystallization experiments of I‐CvuI in complex with its DNA target in the presence of Mg 2+ yielded crystals suitable for X‐ray diffraction analysis. The crystals belonged to the orthorhombic space group P 2 1 2 1 2 1 , with unit‐cell parameters a = 62.83, b = 83.56, c = 94.40 Å. The self‐rotation function and the Matthews coefficient suggested the presence of one protein–DNA complex per asymmetric unit. The crystals diffracted to a resolution limit of 1.9 Å using synchrotron radiation.