Annealing helicase HARP closes RPA-stabilized DNA bubbles non-processively.
Nucleic Acids Res
; 45(8): 4687-4695, 2017 05 05.
Article
en En
| MEDLINE
| ID: mdl-28334870
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 â¼600 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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN Superhelicoidal
/
ADN Helicasas
/
Proteína de Replicación A
Límite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2017
Tipo del documento:
Article
País de afiliación:
Países Bajos