Burnham, Daniel R.Nijholt, BasDe Vlaminck, IwijnQuan, JinhuaYusufzai, TimurDekker, Cees2017-06-152017Burnham, Daniel R., Bas Nijholt, Iwijn De Vlaminck, Jinhua Quan, Timur Yusufzai, and Cees Dekker. 2017. “Annealing helicase HARP closes RPA-stabilized DNA bubbles non-processively.” Nucleic Acids Research 45 (8): 4687-4695. doi:10.1093/nar/gkx147. http://dx.doi.org/10.1093/nar/gkx147.http://nrs.harvard.edu/urn-3:HUL.InstRepos:33029868Abstract 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 bubble-closing 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-USJournal Article2017-06-1510.1093/nar/gkx147