The modified nucleosides 2′-deoxy-7-cyano- and 2′-deoxy-7-amido-7-deazaguanosine (dPreQ0 and dADG, respectively) recently discovered in DNA are the products of the bacterial queuosine tRNA modification pathway and the dpd gene cluster, the latter of which encodes proteins that comprise the elaborate Dpd restriction–modification system present in diverse bacteria. Recent genetic studies implicated the dpdA, dpdB and dpdC genes as encoding proteins necessary for DNA modification, with dpdD–dpdK contributing to the restriction phenotype. Here we report the in vitro reconstitution of the Dpd modification machinery from Salmonella enterica serovar Montevideo, the elucidation of the roles of each protein and the X-ray crystal structure of DpdA supported by small-angle X-ray scattering analysis of DpdA and DpdB, the former bound to DNA. While the homology of DpdA with the tRNA-dependent tRNA-guanine transglycosylase enzymes (TGT) in the queuosine pathway suggested a similar transglycosylase activity responsible for the exchange of a guanine base in the DNA for 7-cyano-7-deazaguanine (preQ0), we demonstrate an unexpected ATPase activity in DpdB necessary for insertion of preQ0 into DNA, and identify several catalytically essential active site residues in DpdA involved in the transglycosylation reaction. Further, we identify a modification site for DpdA activity and demonstrate that DpdC functions independently of DpdA/B in converting preQ0-modified DNA to ADG-modified DNA.

中文翻译：

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DNA 中的 7-脱氮鸟嘌呤：DNA 修饰系统的功能和结构解析

最近在 DNA 中发现的修饰核苷 2'-deoxy-7-cyano- 和 2'-deoxy-7-amido-7-deazaguanosine（分别为 dPreQ0 和 dADG）是细菌队列 tRNA 修饰途径和 dpd 基因的产物簇，后者编码的蛋白质构成了存在于不同细菌中的复杂 Dpd 限制修饰系统。最近的遗传学研究表明 dpdA、dpdB 和 dpdC 基因是 DNA 修饰所必需的编码蛋白，其中 dpdD–dpdK 有助于限制表型。在这里，我们报告了蒙得维的亚沙门氏菌血清型 Dpd 修饰机制的体外重组，阐明了每种蛋白质的作用以及 DpdA 和 DpdB 的小角度 X 射线散射分析支持的 DpdA 的 X 射线晶体结构，前者与 DNA 结合。虽然 DpdA 与 queuosine 途径中 tRNA 依赖性 tRNA-鸟嘌呤转糖基酶 (TGT) 的同源性表明类似的转糖基酶活性负责将 DNA 中的鸟嘌呤碱基交换为 7-cyano-7-deazaguanine (preQ0)，我们证明了 DpdB 中意外的 ATPase 活性是将 preQ0 插入 DNA 所必需的，并确定了 DpdA 中参与转糖基化反应的几个催化必需活性位点残基。此外，我们确定了 DpdA 活动的修饰位点，并证明 DpdC 在将 preQ0 修饰的 DNA 转化为 ADG 修饰的 DNA 时独立于 DpdA/B 发挥作用。