Mutational analysis of the CG recognizing DNA methyltransferase SssI: Insight into enzyme-DNA interactions
Mutational analysis of the CG recognizing DNA methyltransferase SssI: Insight into enzyme-DNA interactions
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DOI：
10.1016/j.bbapap.2009.07.016
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发表时间：
2009-11-01
影响因子：
3.2
通讯作者：
Gromova, Elizaveta S.
中科院分区：
文献类型：
--
作者：
Darii, Maria V.;Cherepanova, Natalia A.;Gromova, Elizaveta S.
为了表征 M.SssI 进行 DNA 甲基化的重要步骤，M.SssI 是一种原核 DNA-(胞嘧啶 C5) 甲基转移酶 (C5-MTase)，具有真核 C5-MTase (5'-CG-3') 的特异性，根据序列比对和计算模型选择了 10 个氨基酸，进行了突变分析。研究了野生型和突变型 M.SssI 变体，以确定甲基化活性、DNA 结合亲和力、诱导碱基翻转的能力以及与含有基于机制的抑制剂 2-嘧啶酮的 DNA 底物形成共价复合物的能力。当野生型 M.SssI 与含有 2-氨基嘌呤代替目标胞嘧啶的底物 DNA 结合时，会诱导强荧光，表明目标碱基发生翻转。荧光减少、甲基转移酶活性中度或剧烈丧失以及 DNA 结合减少表明保守的 5745（基序 IV）、8232（基序 VIII，bar 下的 QxRx (R) 和 T313（可变区，barL 下的保守 (T)）以及非保守的 Q147 参与碱基翻转。E186 的替换（基序 VI，(E)） NV 下的基序 VIII，xR 下的 Qx (R) 与丙氨酸导致甲基转移酶活性丧失，但不损害 DNA 结合亲和力。这些数据与 E186 和 8230 的催化作用一致，并首次为 C5-MTase (C) 2009 中迄今为止未研究的基序 VIII 的不变精氨酸的基本功能提供了实验支持。 Elsevier B.V. 保留所有权利。
To characterize important steps of DNA methylation by M.SssI, a prokaryotic DNA-(cytosine C5)methyltransferase (C5-MTase) sharing the specificity of eukaryotic C5-MTases (5'-CG-3'), ten amino acids, selected on the basis of sequence alignments and a computational model, were subjected to mutational analysis. Wild-type and mutant M.SssI variants were studied to determine methylation activity, DNA binding affinity, capacity to induce base flipping, and ability to form covalent complex with a DNA substrate containing the mechanism-based inhibitor 2-pyrimidinone. Wild-type M.SssI induced strong fluorescence when bound to substrate DNA containing 2-aminopurine in place of the target cytosine, indicating flipping of the target base. Reduced fluorescence, moderate, or drastic loss of methyltransferase activity and reduced DNA binding suggest the involvement of the conserved 5745 (motif IV), 8232 (motif VIII, QxRx (R) under bar, and T313 (variable region, conserved (T) under barL), as well as of the non-conserved Q147 in base flipping. Replacement of E186 (motif VI, (E) under bar NV) and 8230 (motif VIII, Qx (R) under bar xR) with alanine resulted in loss of methyltransferase activity without impairing DNA binding affinity. These data are consistent with the catalytic role of E186 and 8230, and provide, for the first time, experimental support for the essential function of the hitherto not investigated invariant arginine of motif VIII in C5-MTases. (C) 2009 Elsevier B.V. All rights reserved.