Force Regulated Association Dynamics of RPA on Forked DNA

Abstract

Replication protein A (RPA) is involved in virtually all aspects of eukaryotic DNA processing, including replication, recombination and repair. The strong binding of RPA to single-stranded DNA (ssDNA), which provides protection from nucleolytic cleavage, is well established. Binding to partially double-stranded DNA (dsDNA) and disruption of DNA intermediates possessing secondary structures was reported previously, however the underlying mechanism remains unclear.Utilizing single-molecule magnetic tweezers, we now show that both human and yeast RPA can open a DNA hairpin subjected to force. For this well-defined substrate geometry which closely mimics a replication fork, we measured the force-dependent RPA association and dissociation, whilst resolving individual binding events.To explain the observed helix opening activity, we propose a passive model: Transient openings of the DNA helix, enhanced by stronger forces, become trapped by RPA binding. When the force is reduced, dissociation is driven by helix rehybridization. This leads to an exponential dependence of the association and dissociation rates on the applied force, in agreement with our data.By extrapolating our measured rates, we conjecture that RPA does not disrupt dsDNA on its own in the absence of force, but rather seems to rely on the active unwinding of a helicase. RPA could then strongly bind and protect ssDNA produced in its wake, providing a scaffold for the recruitment of further DNA processing enzymes. While the fork is held open, changes to the force exerted by downstream enzymes may regulate the degree of RPA binding on the ssDNA. Upon completion of the DNA processing step the fork would become unblocked. Rapid helix rehybridization then expels RPA from the ssDNA with dissociation rates on the order of hundreds of base-pairs per second, such that the completely processed dsDNA is returned to its native RPA-unbound state.