TY - JOUR
T1 - Hhal methyltransferase flips its target base out of the DNA helix
AU - Klimasauskas, Saulius
AU - Kumar, Sanjay
AU - Roberts, Richard J.
AU - Cheng, Xiaodong
N1 - Funding Information:
Reprint requests should be addressed to X. C. The Biology Department single-crystal diffraction facility of Brookhaven National Laboratory at beamline Xl 2-C of the National Synchrotron Light Source is supported by United States Department of Energy, Office of Health and Environmental Research. It is a pleasure to acknowledge R. M. Sweet and V. Stojanoff for valuable assistance with the data collection and for generous access to the X12-C beamline, and C. L. Lawson for the modified version of TRNSUM for translation solution. We thank K. McCloy for technical assistance in the purification and crystallization; R. Knott for oligonucleotides; C. Lin for computational help; J. W. Pflugrath, D. Barford, R. Xu, X. Zhang, and S. S. Smith for helpful discussions. This work was supported by grants from National Institutes of Health GM49245 to X. C. and GM46127 to R. J. R.
PY - 1994/1/28
Y1 - 1994/1/28
N2 - The crystal structure has been determined at 2.8 Å resolution for a chemically-trapped covalent reaction intermediate between the Hhal DNA cytosine-5-methyltransferase, S-adenosyl-l-homocysteine, and a duplex 13-mer DNA oligonucleotide containing methylated 5-fluorocytosine at its target. The DNA is located in a cleft between the two domains of the protein and has the characteristic conformation of B-form DNA, except for a disrupted G-C base pair that contains the target cytosine. The cytosine residue has swung completely out of the DNA helix and is positioned in the active site, which itself has undergone a large conformational change. The DNA is contacted from both the major and the minor grooves, but almost all base-specific interactions between the enzyme and the recognition bases occur in the major groove, through two glycine-rich loops from the small domain. The structure suggests how the active nucleophile reaches its target, directly supports the proposed mechanism for cytosine-5 DNA methylation, and illustrates a novel mode of sequence-specific DNA recognition.
AB - The crystal structure has been determined at 2.8 Å resolution for a chemically-trapped covalent reaction intermediate between the Hhal DNA cytosine-5-methyltransferase, S-adenosyl-l-homocysteine, and a duplex 13-mer DNA oligonucleotide containing methylated 5-fluorocytosine at its target. The DNA is located in a cleft between the two domains of the protein and has the characteristic conformation of B-form DNA, except for a disrupted G-C base pair that contains the target cytosine. The cytosine residue has swung completely out of the DNA helix and is positioned in the active site, which itself has undergone a large conformational change. The DNA is contacted from both the major and the minor grooves, but almost all base-specific interactions between the enzyme and the recognition bases occur in the major groove, through two glycine-rich loops from the small domain. The structure suggests how the active nucleophile reaches its target, directly supports the proposed mechanism for cytosine-5 DNA methylation, and illustrates a novel mode of sequence-specific DNA recognition.
UR - https://www.scopus.com/pages/publications/0028010888
UR - https://www.scopus.com/pages/publications/0028010888#tab=citedBy
U2 - 10.1016/0092-8674(94)90342-5
DO - 10.1016/0092-8674(94)90342-5
M3 - Article
C2 - 8293469
AN - SCOPUS:0028010888
SN - 0092-8674
VL - 76
SP - 357
EP - 369
JO - Cell
JF - Cell
IS - 2
ER -