Restriction enzyme BsoBI-DNA complex: a tunnel for recognition of degenerate DNA sequences and potential histidine catalysis.
BACKGROUND
Restriction endonucleases form a diverse family of proteins with substantial variation in sequence, structure, and interaction with recognition site DNA. BsoBI is a thermophilic restriction endonuclease that exhibits both base-specific and degenerate recognition within the sequence CPyCGPuG.


RESULTS
The structure of BsoBI complexed to cognate DNA has been determined to 1.7 A resolution, revealing several unprecedented features. Each BsoBI monomer is formed by inserting a helical domain into an expanded EcoRI-type catalytic domain. DNA is completely encircled by a BsoBI dimer. Recognition sequence DNA lies within a 20 A long tunnel of protein that excludes bulk solvent. Interactions with the specific bases are made in both grooves through direct and water-mediated hydrogen bonding. Interaction with the degenerate position is mediated by a purine-specific hydrogen bond to N7, ensuring specificity, and water-mediated H bonding to the purine N6/O6 and pyrimidine N4/O4, allowing degeneracy. In addition to the conserved active site residues of the DX(n)(E/D)ZK restriction enzyme motif, His253 is positioned to act as a general base.


CONCLUSIONS
A catalytic mechanism employing His253 and two metal ions is proposed. If confirmed, this would be the first example of histidine-mediated catalysis in a restriction endonuclease. The structure also provides two novel examples of the role of water in protein-DNA interaction. Degenerate recognition may be mediated by employing water as a hydrogen bond donor or acceptor. The structure of DNA in the tunnel may also be influenced by the absence of bulk solvent.
van der Woerd
M J
MJ
Department of Biological Sciences, Purdue University, 47907, West Lafayette, IN, USA.
Pelletier
J J
JJ
Xu
S
S
Friedman
A M
AM
D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry*; D001411:Geobacillus stearothermophilus / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D006639:Histidine; D004247:DNA; C028678:CYCGRG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific