Chemo-Mechanical Study of a Hexameric Helicase on the Single-Molecule Level

Abstract

Cell division involves DNA replication that requires the opening of double stranded DNA by cellular machines known as helicases. Although many of those essential enzymes are of hexameric structure, not much is known about coordination and the mechano-chemical function of these multimeric enzymes. Here, we study with single-molecule FRET (Forster resonance energy transfer) a hexameric helicase of the DnaB family, called G40P. DnaB helicase is the essential replication helicase in prokaryotes and consists of 6 identical subunits that exhibit the widely shared RecA-fold in biological enzymes. In order to gain information about the chemo-mechanical cycle of G40P, we followed the time trajectory of individual enzymes while unwinding a DNA duplex. By the addition of the non-hydrolyzable ATP analogue ATPgS to the reaction at low ratio of ATPgS to ATP, we observed significant stalls during the unwinding process. Varying the concentration of ATPgS did not affect the lifetime of the stall, which indicates a strong coordination between the identical subunits. Based on this observation, we propose a highly coordinated subsequent ATP hydrolysis between the subunits, where binding ATPgS at a single site can stall the entire helicase. Furthermore, under suboptimal conditions like low ATP concentrations, we observed frequent repetitive slippage events of individual helicases, indicating a transient loss of tight binding to the DNA substrate.