Annealing helicase HARP closes RPA-stabilized DNA bubbles non-processively
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Burnham, Daniel R.
Nijholt, Bas
De Vlaminck, Iwijn
Quan, Jinhua
Dekker, Cees
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https://doi.org/10.1093/nar/gkx147Metadata
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Burnham, 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.Abstract
Abstract 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.Other Sources
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5416776/pdf/Terms of Use
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