TY - JOUR
T1 - Mechanistic insights from the structures of HincII bound to cognate DNA cleaved from addition of Mg2+ and Mn2+
AU - Etzkorn, Christopher
AU - Horton, Nancy C.
N1 - Funding Information:
Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences. This work was funded by NIH 066805-01A1.
PY - 2004/10/29
Y1 - 2004/10/29
N2 - The three-dimensional X-ray crystal structures of HincII bound to cognate DNA containing GTCGAC and Mn2+ or Mg2+, at 2.50 Å and 2.95 Å resolution, respectively, are presented. In both structures, the DNA is found cleaved, and the positions of the active-site groups, cleaved phosphate group, and 3′ oxygen atom of the leaving group are in very similar positions. Two highly occupied Mn2+ positions are found in each active site of the four crystallographically independent subunit copies in the HincII/DNA/Mn2+ structure. The manganese ion closest to the previously identified single Ca2+ position of HincII is shifted 1.7 Å and has lost direct ligation to the active-site aspartate residue, Asp127. A Mn2+-ligated water molecule in a position analogous to that seen in the HincII/DNA/Ca2+ structure, and proposed to be the attacking nucleophile, is beyond hydrogen bonding distance from the active-site lysine residue, Lys129, but remains within hydrogen bonding distance from the proRp oxygen atom of the phosphate group 3′ to the scissile phosphate group. In addition, the position of the cleaved phosphate group is on the opposite side of the axis connecting the two metal ions relative to that found in the BamHI/product DNA/Mn2+ structure. Mechanistic implications are discussed, and a model for the two-metal-ion mechanism of DNA cleavage by HincII is proposed.
AB - The three-dimensional X-ray crystal structures of HincII bound to cognate DNA containing GTCGAC and Mn2+ or Mg2+, at 2.50 Å and 2.95 Å resolution, respectively, are presented. In both structures, the DNA is found cleaved, and the positions of the active-site groups, cleaved phosphate group, and 3′ oxygen atom of the leaving group are in very similar positions. Two highly occupied Mn2+ positions are found in each active site of the four crystallographically independent subunit copies in the HincII/DNA/Mn2+ structure. The manganese ion closest to the previously identified single Ca2+ position of HincII is shifted 1.7 Å and has lost direct ligation to the active-site aspartate residue, Asp127. A Mn2+-ligated water molecule in a position analogous to that seen in the HincII/DNA/Ca2+ structure, and proposed to be the attacking nucleophile, is beyond hydrogen bonding distance from the active-site lysine residue, Lys129, but remains within hydrogen bonding distance from the proRp oxygen atom of the phosphate group 3′ to the scissile phosphate group. In addition, the position of the cleaved phosphate group is on the opposite side of the axis connecting the two metal ions relative to that found in the BamHI/product DNA/Mn2+ structure. Mechanistic implications are discussed, and a model for the two-metal-ion mechanism of DNA cleavage by HincII is proposed.
KW - metal ion catalysis
KW - phosphoryl transfer
KW - protein-nucleic acid recognition
KW - restriction enzyme
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U2 - 10.1016/j.jmb.2004.08.082
DO - 10.1016/j.jmb.2004.08.082
M3 - Article
C2 - 15476804
AN - SCOPUS:5144231405
VL - 343
SP - 833
EP - 849
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 4
ER -