Torsional regulation of hRPA-induced unwinding of double-stranded DNA |
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| Authors: | Iwijn De Vlaminck Iztok Vidic Marijn T J van Loenhout Roland Kanaar Joyce H G Lebbink Cees Dekker |
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| Institution: | 1.Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, 2.Department of Cell Biology and Genetics, Cancer Genomic Center and 3.Department of Radiation Oncology, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands |
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| Abstract: | 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. |
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