Structure and substrate recognition of the Escherichia coli DNA adenine methyltransferase
Structure and substrate recognition of the Escherichia coli DNA adenine methyltransferase
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DOI：
10.1016/j.jmb.2006.02.028
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发表时间：
2006-04-28
影响因子：
5.6
通讯作者：
Cheng, XD
中科院分区：
文献类型：
--
作者：
Horton, JR;Liebert, K;Cheng, XD
在s -腺苷- l-同型半胱氨酸存在的情况下，以1.89 (A)过环分辨率测定了大肠杆菌坝DNA-（腺嘌呤- n6）-甲基转移酶与同源DNA复合物的结构。通过定点诱变、DNA甲基化动力学和荧光停流实验研究了DNA识别和碱基翻转动力学。我们的数据说明了DNA识别和碱基翻转耦合的机制。在GATC位点的第二（3’）半部分，与非目标链的接触是由R124与第四个碱基对建立的，由L122和P134与第三个碱基对建立的。Y119的芳香环插入到DNA的第二和第三碱基对之间，这是碱基翻转发生的必要条件。与先前发表的噬菌体T4坝的结构相比，在大肠杆菌坝中进行了三个主要的新观察。(1)第一个Gua被K9识别，去除K9将取消第一个碱基对识别。(2)在没有s -腺苷- l-蛋氨酸的情况下，翻转的靶标Ade与EcoDam表面结合，这说明了在碱基翻转途径中可能存在一种中间体。(3)孤立的Thy残基在与N120接触时采用螺旋外或螺旋内位置，表现出结构灵活性。(c) 2006 Elsevier Ltd.版权所有。
The structure of the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase in complex with cognate DNA was determined at 1.89 (A) over circle resolution in the presence of S-adenosyl-L-homocysteine. DNA recognition and the dynamics of base-flipping were studied by site-directed mutagenesis, DNA methylation kinetics and fluorescence stopped-flow experiments. Our data illustrate the mechanism of coupling of DNA recognition and base-flipping. Contacts to the non-target strand in the second (3') half of the GATC site are established by R124 to the fourth base-pair, and by L122 and P134 to the third base-pair. The aromatic ring of Y119 intercalates into the DNA between the second and third base-pairs, which is essential for base-flipping to occur. Compared to previous published structures of bacteriophage T4 Dam, three major new observations are made in E. coli Dam. (1) The first Gua is recognized by K9, removal of which abrogates the first base-pair recognition. (2) The flipped target Ade binds to the surface of EcoDam in the absence of S-adenoSyl-L-methionine, which illustrates a possible intermediate in the base-flipping pathway. (3) The orphaned Thy residue displays structural flexibility by adopting an extrahelical or intrahelical position where it is in contact to N120. (c) 2006 Elsevier Ltd. All rights reserved.