The substrate binding interface of alkylpurine DNA glycosylase AlkD.
The substrate binding interface of alkylpurine DNA glycosylase AlkD.
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DOI：
10.1016/j.dnarep.2013.10.009
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发表时间：
2014-01
期刊：
影响因子：
3.8
通讯作者：
Eichman, Brandt F.
中科院分区：
文献类型：
--
作者：
Mullins, Elwood A.;Rubinson, Emily H.;Eichman, Brandt F.
串联螺旋重复序列已成为一种重要的DNA结合结构。DNA糖基酶AlkD去除N3和N7烷基化的碱基，使用重复的螺旋基序结合双链DNA，并选择性地在非Watson-Crick碱基对上暂停。DNA骨架的重塑促进了损伤和互补碱基分别向溶剂和蛋白质表面的核苷酸翻转。这种新的DNA结合结构的重要特征使AlkD能够在不接触病变的情况下区分受损和正常的DNA，但人们对此知之甚少。在这里，我们通过广泛的突变分析表明，DNA结合和N3-甲基腺嘌呤(3 MA)和N7-甲基鸟嘌呤(7 Mg)的切除取决于DNA结合界面上的每个残基。破坏与DNA骨架的静电或疏水相互作用大大降低了结合亲和力和催化活性。这些结果表明，似乎只参与一般DNA结合的残基对催化活性是重要的，并暗示碱基切除是由这种新的DNA结合结构的整个底物界面提供的结合能驱动的。
Tandem helical repeats have emerged as an important DNA binding architecture. DNA glycosylase AlkD, which excises N3- and N7-alkylated nucleobases, uses repeating helical motifs to bind duplex DNA and to selectively pause at non-Watson-Crick base pairs. Remodeling of the DNA backbone promotes nucleotide flipping of the lesion and the complementary base into the solvent and toward the protein surface, respectively. The important features of this new DNA binding architecture that allow AlkD to distinguish between damaged and normal DNA without contacting the lesion are poorly understood. Here, we show through extensive mutational analysis that DNA binding and N3-methyladenine (3mA) and N7-methylguanine (7mG) excision are dependent upon each residue lining the DNA binding interface. Disrupting electrostatic or hydrophobic interactions with the DNA backbone substantially reduced binding affinity and catalytic activity. These results demonstrate that residues seemingly only involved in general DNA binding are important for catalytic activity and imply that base excision is driven by binding energy provided by the entire substrate interface of this novel DNA binding architecture.
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