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.
Department of Biological Sciences, Purdue University, 47907, West Lafayette, IN, USA.
M J
MJ
van der Woerd
J J
JJ
Pelletier
S
S
Xu
A M
AM
Friedman
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