DNA double-stranded breaks (DSBs) are one of the most deleterious forms of DNA damage resulting in genetic instability and the development of cancer in humans. DSBs are repaired by homologous recombination, during which the DNA ends are resected by a helicase-nuclease to generate 3' single strand tails. In archaea, the repair mechanism requires the Mre11-Rad50 and the ATP-dependent helicase-nuclease HerA-NurA complex. In HerA-NurA, dsDNA enters the hexameric HerA ATPase channel and is subsequently unwound by the narrower dimeric NurA, which acts as a ploughshare. The processes of DNA translocation and unwinding are “fueled” by the binding of six ATPs to the nucleotide-binding sites on HerA monomers. Despite the importance of this system in DSBs repair, the assembly mechanism of HerA-NurA and its interactions with nucleotides remain largely unknown.
Combining mass spectrometry (MS) with biochemical analysis and modelling, we propose a mechanism of HerA-NurA DNA assembly and ATP binding. We performed nucleotide-binding studies to investigate the stability and conformations of the complex upon ATP binding. Our findings suggest an ordered ATP binding mechanism where the presence of dsDNA enables conformational changes that free four ATP binding sites. We further highlight the stabilizing role of NurA, the presence of which imposes oligomeric selectivity on the HerA. Interestingly, we show that the HerA in the absence of NurA predominantly exists as heptamer, while binding of NurA dimer promotes its assembly with the hexameric HerA. We postulate that the switch between heptamer to hexamer may provide a ring-opening mechanism for efficient loading of the double-stranded DNA onto the HerA-NurA. Overall, we believe that this selectivity process may be related to a cellular mechanism of DNA damage recognition and repair, where the more flexible HerA heptamer senses damaged DNA, recruits NurA that enables the formation of HerA-NurA complex for dsDNA translocation and repair.