SEQUENCE-SPECIFIC DNA-BINDING BY THE MSPI DNA METHYLTRANSFERASE
SEQUENCE-SPECIFIC DNA-BINDING BY THE MSPI DNA METHYLTRANSFERASE
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DOI：
10.1093/nar/20.12.3167
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发表时间：
1992-06-25
影响因子：
14.9
通讯作者：
ROBERTS, RJ
中科院分区：
文献类型：
--
作者：
DUBEY, AK;ROBERTS, RJ
MspI甲基转移酶（MspI methyltransferase, MspI）识别序列CCGG，并在第一个c -残基催化5-甲基胞嘧啶的形成。我们利用凝胶迁移位移法和dna印迹法研究了平衡条件下m.m sspi的序列特异性dna结合特性。mspi以序列特异性的方式与DNA结合，既可以单独结合，也可以在正常的甲基供体s -腺苷- l-蛋氨酸以及类似物、丝状真菌素和s -腺苷- l-同型半胱氨酸存在的情况下结合。在s -腺苷- l-同型半胱氨酸存在的情况下，M.Mspl对含有半甲基化识别序列的DNA具有最高的结合亲和力（Kd = 3.6 × 10(-7)M），但与未甲基化DNA的结合较差（Kd = 8.3 × 10(-7)M）。令人惊讶的是，它与完全甲基化的DNA （Kd = 4.2 x 10(-6)M）的结合表现出特异性，尽管很差。在没有辅助因子的情况下，M.Mspl与含有其识别序列CCGG的DNA的结合紧密程度约为非特异性序列的5倍。当存在s -腺苷- l-蛋氨酸、s -腺苷- l-同型半胱氨酸或丝状菌素时，特异性和非特异性序列的区别增加了100倍。DNA足迹研究表明，M.MspI覆盖了16个碱基对的DNA，识别序列CCGG不对称地位于足迹内。
The MspI methyltransferase (M.MspI) recognizes the sequence CCGG and catalyzes the formation of 5-methylcytosine at the first C-residue. We have investigated the sequence-specific DNA-binding properties of M.MspI under equilibrium conditions, using gel-mobility shift assays and DNasel footprinting. M.MspI binds to DNA in a sequence-specific manner either alone or in the presence of the normal methyl donor S-adenosyl-L-methionine as well as the analogues, sinefungin and S-adenosyl-L-homocysteine. In the presence of S-adenosyl-L-homocysteine, M.Mspl shows the highest binding affinity to DNA containing a hemimethylated recognition sequence (Kd = 3.6 x 10(-7)M), but binds less well to unmethylated DNA (Kd = 8.3 x 10(-7)M). Surprisingly it shows specific, although poor, binding to fully methylated DNA (Kd = 4.2 x 10(-6)M). M.Mspl binds approximately 5-fold more tightly to DNA containing its recognition sequence, CCGG, than to nonspecific sequences in the absence of cofactors. In the presence of S-adenosyl-L-methionine, S-adenosyl-L-homocysteine or sinefungin the discrimination between specific and non-specific sequences increases up to 100-fold. DNasel footprinting studies indicate that 16 base pairs of DNA are covered by M.MspI, with the recognition sequence CCGG located asymmetrically within the footprint.