Annealing helicase HARP closes RPA-stabilized DNA bubbles non-processively.
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.
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, 2629 HZ, The Netherlands.
Daniel R
DR
Burnham
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, 2629 HZ, The Netherlands.
Bas
B
Nijholt
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, 2629 HZ, The Netherlands.
Iwijn
I
De Vlaminck
Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02215, USA.
Jinhua
J
Quan
Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02215, USA.
Timur
T
Yusufzai
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, 2629 HZ, The Netherlands.
Cees
C
Dekker
D010582:Bacteriophage lambda / Q000235:genetics / Q000378:metabolism; D001696:Biomechanical Phenomena; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D060526:Magnetic Fields; D052898:Optical Tweezers; D009994:Osmolar Concentration; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A / Q000737:chemistry* / Q000378:metabolism
D004278:DNA, Superhelical; D011994:Recombinant Proteins; D051716:Replication Protein A; C412482:SMARCAL1 protein, human; D004265:DNA Helicases