Structural and biochemical analyses of hemimethylated DNA binding by the SeqA protein
Structural and biochemical analyses of hemimethylated DNA binding by the SeqA protein
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DOI：
10.1093/nar/gkh173
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Yokoyama, S
中科院分区：
文献类型：
--
作者：
Fujikawa, N;Kurumizaka, H;Yokoyama, S
大肠杆菌SeqA蛋白识别染色体oriC区域中的11个半甲基化G-（m）A-T-C位点，并防止在一个细胞周期内复制过度起始。半甲基化DNA的SeqA C-末端结构域的晶体结构揭示了N-6-甲基腺嘌呤识别机制;然而，半甲基化和完全甲基化状态之间的区分机制仍然难以捉摸。在本研究中，我们对具有SeqA的最小DNA结合结构域的半甲基化G-（m）A-T-C序列（SeqA（71 - 181））进行了突变分析，发现SeqA（71 -181）与含有错配（m）A：G碱基对的序列[G-（m）A（：G）-T-C]的半甲基化DNA特异性结合与正常半甲基化G-（m）A（：T）-T-C序列一样有效。我们分别在2.5和3.0埃分辨率下测定了与错配和正常半甲基化DNA复合的SeqA（71-181）的晶体结构，并发现错配的（m）A：G碱基对和正常的（m）A：T碱基对以类似的方式被SeqA识别。此外，在两种晶体结构中，电子密度存在于未甲基化的腺嘌呤附近，其仅在完全甲基化状态下被甲基化。这种电子密度，可能是由于水分子或金属离子，可以存在于半甲基化状态，但不是在完全甲基化状态，因为空间冲突与额外的甲基。
The Escherichia coli SeqA protein recognizes the 11 hemimethylated G-(m)A-T-C sites in the oriC region of the chromosome, and prevents replication over-initiation within one cell cycle. The crystal structure of the SeqA C-terminal domain with hemimethylated DNA revealed the N-6-methyladenine recognition mechanism; however, the mechanism of discrimination between the hemimethylated and fully methylated states has remained elusive. In the present study, we performed mutational analyses of hemimethylated G-(m)A-T-C sequences with the minimal DNA-binding domain of SeqA (SeqA(71-181)), and found that SeqA(71-181) specifically binds to hemimethylated DNA containing a sequence with a mismatched (m)A:G base pair [G-(m)A(:G)-T-C] as efficiently as the normal hemimethylated G-(m)A(:T)-T-C sequence. We determined the crystal structures of SeqA(71-181) complexed with the mismatched and normal hemimethylated DNAs at 2.5 and 3.0 Angstrom resolutions, respectively, and found that the mismatched (m)A:G base pair and the normal (m)A:T base pair are recognized by SeqA in a similar manner. Furthermore, in both crystal structures, an electron density is present near the unmethylated adenine, which is only methylated in the fully methylated state. This electron density, which may be due to a water molecule or a metal ion, can exist in the hemimethylated state, but not in the fully methylated state, because of steric clash with the additional methyl group.