Replication

Helicases are enzymes essential for DNA replication, recombination, and repair. In these biological processes, helicases serve the function of separating (unwinding) the two strands that form the DNA double helix. Helicase unwinds DNA by translocating along one strand, while simultaneously separating the second strand. Hexameric helicases are ubiquitous from phages to humans. The hexameric helicase of bacteriophage T7 is a model helicase for understanding how these types of helicases work to translocate along DNA and separate the strands of duplex DNA.

This cartoon illustrates the experimental configuration for the observation of the unwinding of dsDNA by a single T7 helicase (not to scale). One strand of the dsDNA (purple) to be unwound was attached to a trapped microsphere. The other strand (cyan) was anchored to a microscope coverslip surface. The microsphere was held in a feedback-enhanced optical trap (red) so that its position relative to the trap center and the force on it could be measured. Helicase (yellow) unwinding of dsDNA was monitored as an increase in the ssDNA length.

One intriguing question was how helicase couples its translocation along ssDNA to unwinding of dsDNA. That is, does helicase passively wait for thermal opening of the DNA fork before its forward translocation or will it actively destabilize the fork to facilitate fork opening? We addressed this question by directly probing the kinetics of the motion of individual T7-helicase molecules as they unwound double-stranded DNA (dsDNA) or translocated on single-stranded DNA (ssDNA) (Johnson et al., Cell, 2007). We showed that dsDNA unwinding rate depends on DNA sequence as well as an external force applied to the unwinding fork, demonstrating that the fork acts as a barrier to helicase translocation. Theoretical modeling of the helicase motion suggests this helicase utilizes an active unwinding mechanism by destabilizing the bases near the junction. These results provide insight into the mechanism by which helicase and DNA polymerase work together to efficiently replicate DNA.


Force-velocity measurements during helicase unwinding. (A) Example traces of helicase unwinding of dsDNA at various forces. Unwinding velocity varied dramatically with the applied force on ssDNA. (B) Measured force-velocity relation and its comparison with predictions based on various models. Measured force-velocity relation deviated significantly from a simple passive model at step sizes (δ) 1, 2, or 3 bp; larger δ only made the deviation larger. In contrast, an active model with δ = 2 bp and destabilization energy ΔGd = 1.2 kBT per bp over a 6 bp range agrees well with the measurements. Also marked on the plot are the ssDNA translocation rate k0 and the minimum (critical) DNA mechanical unwinding force Fc for the given sequence. Error bars indicate standard errors of the means.

We also addressed whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation. Previous bulk studies concluded that T7 helicase cannot unwind in the presence of ATP, preferring dTTP as a fuel source. Surprisingly, we found that ATP supported faster unwinding than dTTP, but unwinding was frequently interrupted by helicase slippage. These results resolved the apparent lack of unwinding activity seen in bulk studies with ATP. This discovery provided a window of opportunity for us to investigate how the hexamer’s subunits coordinated their catalysis to maintain high unwinding processivity (Sun et al., Nature, 2011). We found that T7 helicase binds and hydrolyses ATP and dTTP by competitive kinetics, such that the unwinding rate is dictated simply by their respective maximum rates Vmax, Michaelis constants KM, and concentrations. In contrast, processivity does not follow a simple competitive behavior and shows a cooperative dependence on nucleotide concentrations. This supports a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Our data indicate that only one subunit at a time can accept a nucleotide while other subunits are nucleotide-ligated and thus they interact with the DNA to ensure processivity. We speculate that slippage may provide an evolutionary advantage for replication: when dNTP concentrations are low, slippage can slow down helicase to allow its synchronization with a slow-moving DNA polymerase. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding during replication.


ATP-induced helicase slippage reveals highly coordinated subunits. (A) A single molecule assay measured the position of a T7 helicase as it unwound dsDNA. With dTTP, helicase did not slip. However, with ATP, unwinding was interrupted by slippage events which prevented processive unwinding. (B) A cartoon illustrating how nucleotide binding to T7 helicase subunits regulates processivity.