DNA Interaction of the CcrM DNA Methyltransferase: A Mutational and Modeling Study
DNA Interaction of the CcrM DNA Methyltransferase: A Mutational and Modeling Study
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DOI：
10.1002/cbic.201200082
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发表时间：
2012-06-18
期刊：
影响因子：
3.2
通讯作者：
Jeltsch, Albert
中科院分区：
文献类型：
--
作者：
Albu, Razvan F.;Zacharias, Martin;Jeltsch, Albert
新月柄杆菌CcrM是DNA-（腺嘌呤N6）-甲基转移酶，其以高特异性甲基化序列GANTC中的腺嘌呤。为了研究其DNA识别机制，我们使用相关甲基转移酶（M1.MboII，修饰GAAGA）的晶体结构作为起点，并将DNA底物对接到其中以识别接近DNA的蛋白质区域。在CcrM和M1.MboII的比对之后，我们确定了CcrM中包含参与DNA识别的残基的四个候选区域。我们在这些区域内突变了20个氨基酸残基，纯化了CcrM变体，并确定了它们在同源GANTC底物和9个近同源底物上的DNA结合和催化活性，其中每个底物在识别序列中包含单个碱基对取代。总之，我们在两个区域中鉴定了四个残基，其突变导致甲基化活性的强烈（>100倍）降低。我们的数据表明，DNA识别CcrM是一个合作的过程，因为中断的关键接触导致的催化活性的损失，但不放松的特异性。此外，我们发现GANTC序列中第五个碱基对的读数发生了变化，另外两个CcrM变体显示出总体活性的较小降低。在此基础上，并与其他DNA甲基转移酶的相同或相关的识别序列的CcrM的序列比对，我们提出了这两个区域在DNA识别CcrM的作用。
Caulobacter crescentus CcrM is a DNA-(adenine N6)-methyltransferase that methylates adenine in the sequence GANTC with high specificity. To investigate its mechanism of DNA recognition, we used the crystal structure of a related methyltransferase (M1.MboII, which modifies GAAGA) as a starting point, and docked into it a DNA substrate to identify the protein regions that approach the DNA. After alignment of CcrM and M1.MboII, we identified four candidate regions in CcrM to contain residues involved in DNA recognition. We mutated 20 amino acid residues within these regions, purified the CcrM variants, and determined their DNA-binding and catalytic activity on a cognate GANTC substrate and on nine near-cognate substrates, each of which contained a single base-pair substitution in the recognition sequence. Altogether, we identified four residues in two of the regions, mutations of which resulted in a strong (>100-fold) reduction of methylation activity. Our data show that DNA recognition by CcrM is a cooperative process, because disruption of critical contacts led to loss of catalytic activity but not to a relaxation in specificity. In addition, we identified a change in the readout of the fifth base pair in the GANTC sequence with two other CcrM variants that showed smaller reductions in overall activity. Based on this and the sequence alignment of CcrM with other DNA methyltransferases of same or related recognition sequence, we propose roles for these two regions in DNA recognition by CcrM.