Structural and functional studies of the NurA-dAMP-Mn2+ complex

MasterDNA double-stranded breaks (DSBs) are the most cytotoxic forms of DNA damage, which can be lethal or cause chromosomal abnormalities. Homologous recombination (HR) pathway uses a homologous template for accurate repair of DSBs. Generation of the 3’ overhang is a critical event during HR repair of DSBs. The 5’-3’ nuclease, NurA, is present in all known thermophilic archaea. NurA plays an important role in generating 3’ single-stranded DNA during archaeal HR, together with HerA and Mre11-Rad50. But there are not much information about the biochemical and structural aspects of NurA. It is poorly understood how NurA processes 5’ strand of DNA and makes 3’ overhang that binding site of the RadA recombinase. I have determined the crystal structures of apo- and dAMP-Mn2+-bound NurA from Pyrococcus furiousus (PfNurA) to provide the basis for its cleavage mechanism. PfNurA forms a pyramid-shaped dimer containing a large central channel on one side, which becomes narrower towards the peak of the pyramid. The central channel can be used for substrate binding. The structure contains a PIWI domain with high similarity to argonaute, endoV nuclease and RNase H that provides the bases of the 5’-3’ directionality of nuclease activity. The two active sites, each of which contains Mn2+ion(s) and dAMP, are at the corners of the elliptical channel near the flat face of the dimer. The 3’ OH group of the ribose ring is directed toward the channel entrance, explaining the 5’-3’ nuclease activity of PfNurA. From structural and biochemical evidences, I provide a DNA binding and cleavage model for PfNurA