title: Torsional regulation of hRPA-induced unwinding of double-stranded DNA.
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.
authors.affiliation: Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
authors.forename: Iwijn
authors.initials: I
authors.lastname: De Vlaminck
authors.forename: Iztok
authors.initials: I
authors.lastname: Vidic
authors.forename: Marijn T J
authors.initials: MT
authors.lastname: van Loenhout
authors.forename: Roland
authors.initials: R
authors.lastname: Kanaar
authors.forename: Joyce H G
authors.initials: JH
authors.lastname: Lebbink
authors.forename: Cees
authors.initials: C
authors.lastname: Dekker
mesh_terms: D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D051716:Replication Protein A / Q000378:metabolism*; D012965:Sodium Chloride / Q000737:chemistry; D013314:Stress, Mechanical
chemical_list: D004277:DNA, Single-Stranded; D051716:Replication Protein A; D012965:Sodium Chloride; D004247:DNA