Structure and mutagenesis of the DNA modification-dependent restriction endonuclease AspBHI.
Structure and mutagenesis of the DNA modification-dependent restriction endonuclease AspBHI.
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DNA修饰依赖性限制性核酸内切酶ASPBHI的结构和诱变。
DOI：
10.1038/srep04246
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发表时间：
2014-03-07
影响因子：
4.6
通讯作者：
Cheng X
中科院分区：
文献类型：
--
作者：
Horton JR;Nugent RL;Li A;Mabuchi MY;Fomenkov A;Cohen-Karni D;Griggs RM;Zhang X;Wilson GG;Zheng Y;Xu SY;Cheng X
修饰依赖的限制性内切酶AspBHI在(C/T)(C/G)(5mC)N(C/G)(N=任何核苷酸)的双链DNA序列中识别5-甲基胞嘧啶(5mC)，并将两链切割固定距离(N12/N16)3‘至修饰的胞嘧啶。我们测定了均四聚体AspBHI的晶体结构。蛋白质的每个亚基包括两个结构域：N端DNA识别结构域和C端DNA裂解结构域。N-末端结构域在结构上类似于真核生物的SET和环相关(SRA)结构域，后者已知与半甲基化的CpG二核苷酸结合。C末端结构类似于经典的II型限制性内切酶，含有DX20EAK的内切酶催化位点基序。为了了解特定氨基酸如何影响AspBHI识别偏好，我们建立了AspBHI-DNA复合体的同源模型，并通过突变探讨了单个氨基酸的重要性。Ser41和Arg42被预测位于修饰胞嘧啶的DNA小沟5‘。用丙氨酸(S41A)和半胱氨酸(S41C)取代Ser41导致了切割活性改变的突变。所有19个Arg42变异体均导致核酸内切酶活性丧失。
The modification-dependent restriction endonuclease AspBHI recognizes 5-methylcytosine (5mC) in the double-strand DNA sequence context of (C/T)(C/G)(5mC)N(C/G) (N = any nucleotide) and cleaves the two strands a fixed distance (N12/N16) 3′ to the modified cytosine. We determined the crystal structure of the homo-tetrameric AspBHI. Each subunit of the protein comprises two domains: an N-terminal DNA-recognition domain and a C-terminal DNA cleavage domain. The N-terminal domain is structurally similar to the eukaryotic SET and RING-associated (SRA) domain, which is known to bind to a hemi-methylated CpG dinucleotide. The C-terminal domain is structurally similar to classic Type II restriction enzymes and contains the endonuclease catalytic-site motif of DX20EAK. To understand how specific amino acids affect AspBHI recognition preference, we generated a homology model of the AspBHI-DNA complex, and probed the importance of individual amino acids by mutagenesis. Ser41 and Arg42 are predicted to be located in the DNA minor groove 5′ to the modified cytosine. Substitution of Ser41 with alanine (S41A) and cysteine (S41C) resulted in mutants with altered cleavage activity. All 19 Arg42 variants resulted in loss of endonuclease activity.
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影响因子：
14.9
作者：
Orlowski J;Bujnicki JM
通讯作者：
Bujnicki JM
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
II型拓扑的重新审视；结构类别和共同保守的核心。
影响因子：
14.9
作者：
Niv MY;Ripoll DR;Vila JA;Liwo A;Vanamee ES;Aggarwal AK;Weinstein H;Scheraga HA
通讯作者：
Scheraga HA
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH
影响因子：
5.6
作者：
OGara, M;Klimasauskas, S;Cheng, XD
通讯作者：
Cheng, XD