Klimasauskas S, Kumar S, Roberts R J, Cheng X
W. M. Keck Structural Biology Laboratory, Cold Spring Harbor, New York 11724.
Cell. 1994 Jan 28;76(2):357-69. doi: 10.1016/0092-8674(94)90342-5.
The crystal structure has been determined at 2.8 A resolution for a chemically-trapped covalent reaction intermediate between the HhaI 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.
已在2.8埃分辨率下确定了HhaI DNA胞嘧啶-5-甲基转移酶、S-腺苷-L-高半胱氨酸与在其靶位点含有甲基化5-氟胞嘧啶的双链13聚体DNA寡核苷酸之间化学捕获的共价反应中间体的晶体结构。DNA位于蛋白质两个结构域之间的裂隙中，具有B型DNA的特征构象，但含有靶标胞嘧啶的G-C碱基对被破坏。胞嘧啶残基已完全从DNA螺旋中摆动出来并定位在活性位点，活性位点本身也发生了很大的构象变化。DNA从大沟和小沟两侧与之接触，但酶与识别碱基之间几乎所有的碱基特异性相互作用都通过小结构域中富含甘氨酸的两个环在大沟中发生。该结构揭示了活性亲核试剂如何到达其靶标，直接支持了所提出的胞嘧啶-5 DNA甲基化机制，并阐明了一种序列特异性DNA识别的新模式。
