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

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.