ATP Hydrolysis Gates an Ultra-Rapid Unwinding Mode of AfXPB Revealed by DNA Electrochemistry

DNA helicases play a central role in DNA metabolism, enabling recombination, replication, transcription, translation, and repair. Recently, an unexpectedly rapid, exponential ATP-stimulated DNA unwinding rate was observed with an Archaeoglobus fulgidus helicase (AfXPB), markedly faster than that of conventional helicases, yet the mechanistic basis remains unresolved. Here, we distinguish the roles of ATP-helicase binding and ATP hydrolysis on the dynamic DNA unwinding activity of AfXPB via an electrochemical assay with redox-active DNA monolayers. To distinguish ATP-helicase binding from ATP hydrolysis, we employ a hydrolysis-resistant analog of ATP, adenylyl imidodiphosphate (AMP-PNP). AMP-PNP suppresses rapid unwinding kinetics, whereas ATP facilitates rapid AfXPB activity. AMP-PNP suppresses signal loss associated with helicase activity for several minutes, then instigates a slow exponential loss consistent with conventional helicase activity. Treating with ATP following AMP-PNP reproduces the characteristic fast-decay, recovery, and slow-decay kinetic sequence observed in ATP-only experiments, with slower rates for each mode. Importantly, this study demonstrates that the ATPase cycle is key to AfXPB's rapid DNA-unwinding kinetics, informing the steps required to unlock its unique biological capabilities.