All cellular single-stranded (ss) DNA is rapidly bound and stabilized by single stranded DNA-binding proteins (SSBs). Replication protein A, the main eukaryotic SSB, is able to unwind double-stranded (ds) DNA by binding and stabilizing transiently forming bubbles of ssDNA. Here, we study the dynamics of human RPA (hRPA) activity on topologically constrained dsDNA with single-molecule magnetic tweezers. We find that the hRPA unwinding rate is exponentially dependent on torsion present in the DNA. The unwinding reaction is self-limiting, ultimately removing the driving torsional stress. The process can easily be reverted: release of tension or the application of a rewinding torque leads to protein dissociation and helix rewinding. Based on the force and salt dependence of the in vitro kinetics we anticipate that the unwinding reaction occurs frequently in vivo. We propose that the hRPA unwinding reaction serves to protect and stabilize the dsDNA when it is structurally destabilized by mechanical stresses.@en

We investigate the mechanistic nature of the Snf2 family protein HARP, mutations of which are responsible for Schimke immuno-osseous dysplasia. Using a single-molecule magnetic tweezers assay, we construct RPA-stabilized DNA bubbles within torsionally constrained DNA to investigate the annealing action of HARP on a physiologically relevant substrate. We find that HARP closes RPA-stabilized bubbles in a slow reaction, taking on the order of tens of minutes for ∼600 bp of DNA to be re-annealed. The data indicate that DNA re-anneals through the removal of RPA, which is observed as clear steps in the bubbleclosing traces. The dependence of the closing rate on both ionic strength and HARP concentration indicates that removal of RPA occurs via an association-dissociation mechanism where HARP does not remain associated with the DNA. The enzyme exhibits classical Michaelis-Menten kinetics and acts cooperatively with a Hill coefficient of 3 ± 1. Our work also allows the determination of some important features of RPA-bubble structures at low supercoiling, including the existence of multiple bubbles and that RPA molecules are mis-registered on the two strands.@en