Annealing helicase HARP closes RPA-stabilized DNA bubbles non-processively.

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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Burnham DR,Nijholt B,De Vlaminck I,Quan J,Yusufzai T,Dekker C

doi

10.1093/nar/gkx147

subject

Has Abstract

pub_date

2017-05-05 00:00:00

pages

4687-4695

issue

8

eissn

0305-1048

issn

1362-4962

pii

3057348

journal_volume

45

pub_type

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