7-deazaguanines in DNA: mechanism and structure of complex genome modification

摘要酶促核苷酸修饰是核酸修饰的关键组成部分， 处理，并用于所有生物体，以解决多种基本需求， 信息传递系统，包括DNA保护，翻译保真度，RNA稳定化， 和表观遗传调控。在RNA和DNA之间的一个显著的例子中， 处理，我们最近发现，已知的最复杂的修改系统之一， 发生在RNA中，负责7-脱氮鸟嘌呤修饰的腺苷（Q）和 古菌素（G+）也被多种生物体用于DNA的修饰。事实上， 细菌由大约10种蛋白质组成一个复杂的限制修饰系统 基于2 ′-脱氧-7-氰基-和2 ′-脱氧-7-氨基-7-脱氮鸟苷（dPreQ 0）的形成， 和dADG），以及这些核苷，以及dG+和2 '-脱氧-7- 氨甲基-7-脱氮鸟苷（dPreQ 1）也已在噬菌体的DNA中发现， 古细菌病毒在确定了参与7- 基于脱氮鸟嘌呤的DNA修饰，我们现在建议阐明分子基础 因为它们的功能。具体目标1和2中所述的研究涉及修改 蛋白质DpdA，DpdB和DpdC，它们共同负责细菌中的机器， dPreQ 0和dADG的形成，并用作基于7-脱氮鸟嘌呤的修饰的模型 在DNA中。在具体目标3中，我们更广泛地研究DpdA家族，并考虑 缺乏DpdB并含有DpdAC融合物，以及噬菌体DpdA，以便更好地理解 在细菌修饰中需要DpdB的隐蔽ATP酶活性， 在细菌和病毒系统中的序列特异性。

SUMMARY. Enzymatically-directed nucleotide modification is a key component of nucleic acid processing and is used by all organisms to address a multitude of fundamental needs in information transfer systems, including protection of DNA, translational fidelity, RNA stabilization, and epigenetic regulation. In a remarkable example of the cross-talk between RNA and DNA processing, we recently discovered that one of the most complex modification systems known to occur in RNA, that responsible for the 7-deazaguanine modifications queuosine (Q) and archaeosine (G+), is also utilized by diverse organisms for the modification of DNA. Indeed, in Bacteria a set of roughly 10 proteins comprise an elaborate restriction-modification (RM) system based on the formation of 2’-deoxy-7-cyano- and 2’-deoxy-7-amido-7-deazaguanosine (dPreQ0 and dADG, respectively) in DNA, and these nucleosides, as well as dG+ and 2’-deoxy-7- aminomethyl-7-deazaguanosine (dPreQ1), have also been found in the DNA of phage and archaeal viruses. Having identified the relevant proteins involved in the formation of 7- deazaguanine based DNA modifications, we are now proposing to elucidate the molecular basis for their function. The research described in specific aims 1 & 2 address the modification machinery in bacteria, the proteins DpdA, DpdB, and DpdC, which together are responsible for the formation of dPreQ0 and dADG, and serve as a model for 7-deazaguanine based modification in DNA. In specific aim 3 we take a broader look at the DpdA family and consider systems that lack DpdB and contain DpdAC fusions, as well as a phage DpdA in order to better understand the need for the cryptic ATPase activity of DpdB in bacterial modification, and the structural basis of sequence specificity in the bacterial and viral systems.