Crystallization and preliminary X-ray analysis of restriction endonuclease FokI bound to DNA.
FokI is a type IIs restriction endonuclease which recognizes an asymmetric DNA sequence and cleaves DNA a short distance away from the sequence. The enzyme is bipartite in nature with its DNA recognition and cleavage functions located on distinct domains. We report here cocrystals of the complete FokI enzyme (579 amino acids) bound to a 20-bp DNA fragment containing its recognition sequence. The complex is amongst the largest protein-DNA complexes to be crystallized, and required macroseeding techniques for optimal crystal growth. The cocrystals diffract to at least 2.8 A in resolution and belong to space group P2(1) with unit cell dimensions of a=67.9 A, b=119.8 A, c=69.1 A, beta = 96.6 degrees. Using specific amino acid analysis we show that asymmetric unit contains a single FokI molecule bound to the 20-bp DNA fragment. This paper reports the first cocrystals of a type IIs restriction endonuclease.
Hirsch
J A
JA
Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
Wah
D A
DA
Dorner
L F
LF
Schildkraut
I
I
Aggarwal
A K
AK
D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism*; D053938:DNA Fragmentation; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific