HHAL METHYLTRANSFERASE FLIPS ITS TARGET BASE OUT OF THE DNA HELIX
HHAL METHYLTRANSFERASE FLIPS ITS TARGET BASE OUT OF THE DNA HELIX
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DOI：
10.1016/0092-8674(94)90342-5
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发表时间：
1994-01-28
期刊：
影响因子：
64.5
通讯作者：
CHENG, XD
中科院分区：
文献类型：
--
作者：
KLIMASAUSKAS, S;KUMAR, S;CHENG, XD
在2.8 A分辨率下测定了HhaI DNA胞嘧啶-1甲基转移酶、S-腺苷+高半胱氨酸和在其靶处含有甲基化5-氟胞嘧啶的双链体1-mer DNA寡核苷酸之间的化学捕获共价反应中间体的晶体结构。DNA位于蛋白质的两个结构域之间的裂缝中，具有B型DNA的特征构象，除了含有靶胞嘧啶的破坏的GC碱基对。胞嘧啶残基已经完全从DNA螺旋中摆动出来，并位于活性位点，其本身已经经历了很大的构象变化。DNA从大沟和小沟接触，但几乎所有的酶和识别碱基之间的碱基特异性相互作用都发生在大沟中，通过两个富含甘氨酸的环从小结构域。该结构表明了活性亲核试剂如何到达其靶标，直接支持胞嘧啶-5 DNA甲基化的拟议机制，并说明了序列特异性DNA识别的新模式。
The crystal structure has been determined at 2.8 A resolution for a chemically-trapped covalent reaction intermediate between the Hhal DNA cytosine-lmethyltransferase, S-adenosyl+ homocysteine, and a duplex l&mer DNA oligonucleotide containing methylated 5fluorocytosine 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 GC 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 basespecific 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.