Annealing Helicase HARP: A Single Molecule Study

Abstract

HepA related protein (HARP) is an annealing helicase capable of rewinding complementary ssDNA stabilized by the single stranded (ss) DNA-binding protein Replication Protein A (RPA) [1]. Mutations in HARP ATPase are responsible for Schimke immuno-osseous dysplasia (SIOD). Recent reports show that HARP is recruited to sites of DNA damage via a binding interaction with RPA and, put HARP functionally at the replication fork under circumstances of replicative stress [2].Here, we use a magnetic-tweezers single-molecule assay to study mechanistic aspects of HARP annealing-helicase activity. In a first step of the single-molecule assay, hRPA-stabilized bubbles of ssDNA are formed in a dsDNA molecule via hRPA-induced unwinding of dsDNA. hRPA locally unwinds double stranded DNA by binding and stabilizing bubbles of ssDNA, a process that relies on torsional energy present in the DNA [3]. Subsequently, we use the hRPA-stabilized bubbles of ssDNA as a starting substrate for studying the annealing helicase activity of HARP. We find that HARP is capable of complete rewinding of hRPA-bound bubbles of ssDNA. We confirm that the annealing helicase activity is ATP-dependent. The recorded reaction rates are proportional to [HARP] and sensitive to [KCL] with slower reactions occurring at higher [KCl]. Our results suggest that HARP, rather than actively displacing hRPA, functions as a ratchet that displaces hRPA by preventing rebinding of dissociated hRPA DNA-binding domains.[1] T. Yusufzai et al. Science (2008).[2] R. Driscoll et al Genes & Dev (2009).[3] I. De Vlaminck et al., Nucleic Acids Research (2010).