Time-resolved fluorescence of 2-aminopurine in DNA duplexes in the presence of the EcoP15I Type III restriction-modification enzyme.
Time-resolved fluorescence of 2-aminopurine in DNA duplexes in the presence of the EcoP15I Type III restriction-modification enzyme.
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DOI：
10.1016/j.bbrc.2014.04.162
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发表时间：
2014-06
影响因子：
3.1
通讯作者：
Long Ma;Xiaohua Wu;G. Wilson;Anita C Jones;D. Dryden
中科院分区：
文献类型：
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作者：
Long Ma;Xiaohua Wu;G. Wilson;Anita C Jones;D. Dryden
EcoP15I 是大肠杆菌的 III 型 DNA 限制和修饰酶。我们证明它包含两个用于 DNA 序列特异性甲基化的修饰 (Mod) 亚基和一个限制性内切酶 (Res) 亚基副本，用于切割含有未甲基化靶序列的 DNA。此前，在存在辅因子的情况下，Mod2二聚体被证明可以使用核苷酸翻转来获得甲基化目标的腺嘌呤碱基（Reddy 和 Rao，J. Mol. Biol. 298 (2000) 597-610.）。令人惊讶的是，Mod2 酶似乎还翻转了靶序列中的第二个腺嘌呤，该腺嘌呤未被甲基化。我们使用腺嘌呤类似物 2-氨基嘌呤的荧光寿命测量表明，只有可甲基化腺嘌呤才会被完整的 Res1Mod2 酶翻转，而且即使在没有辅因子的情况下也会发生这种情况。我们认为这是由于 Res 亚基激活 Mod2core 所致。
EcoP15I is a Type III DNA restriction and modification enzyme ofEscherichia coli. We show that it contains two modification (Mod) subunits for sequence-specific methylation of DNA and one copy of a restriction endonuclease (Res) subunit for cleavage of DNA containing unmethylated target sequences. Previously the Mod2dimer in the presence of cofactors was shown to use nucleotide flipping to gain access to the adenine base targeted for methylation (Reddy and Rao, J. Mol. Biol. 298 (2000) 597–610.). Surprisingly the Mod2enzyme also appeared to flip a second adenine in the target sequence, one which was not subject to methylation. We show using fluorescence lifetime measurements of the adenine analogue, 2-aminopurine, that only the methylatable adenine undergoes flipping by the complete Res1Mod2enzyme and that this occurs even in the absence of cofactors. We suggest that this is due to activation of the Mod2core by the Res subunit.