Active Control of Repetitive Structural Transitions between Replication Forks and Holliday Junctions by Werner Syndrome Helicase

Soochul Shin; Jinwoo Lee; Sangwoon Yoo; Tomasz Kulikowicz; Vilhelm A Bohr; Byungchan Ahn; Sungchul Hohng

doi:10.1016/j.str.2016.06.004

Active Control of Repetitive Structural Transitions between Replication Forks and Holliday Junctions by Werner Syndrome Helicase

Structure. 2016 Aug 2;24(8):1292-1300. doi: 10.1016/j.str.2016.06.004. Epub 2016 Jul 14.

Authors

Soochul Shin¹, Jinwoo Lee¹, Sangwoon Yoo¹, Tomasz Kulikowicz², Vilhelm A Bohr², Byungchan Ahn³, Sungchul Hohng⁴

Affiliations

¹ Department of Physics and Astronomy, Seoul National University, Seoul 08826, Republic of Korea; Institute of Applied Physics, Seoul National University, Seoul 08826, Republic of Korea; National Center for Creative Research Initiatives, Seoul National University, Seoul 08826, Republic of Korea.
² Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
³ Department of Life Sciences, University of Ulsan, Ulsan 680-749, Republic of Korea. Electronic address: bbccahn@mail.ulsan.ac.kr.
⁴ Department of Physics and Astronomy, Seoul National University, Seoul 08826, Republic of Korea; Institute of Applied Physics, Seoul National University, Seoul 08826, Republic of Korea; National Center for Creative Research Initiatives, Seoul National University, Seoul 08826, Republic of Korea; Department of Biophysics and Chemical Biology, Seoul National University, Seoul 08826, Republic of Korea. Electronic address: shohng@snu.ac.kr.

Abstract

The reactivation of stalled DNA replication via fork regression invokes Holliday junction formation, branch migration, and the recovery of the replication fork after DNA repair or error-free DNA synthesis. The coordination mechanism for these DNA structural transitions by molecular motors, however, remains unclear. Here we perform single-molecule fluorescence experiments with Werner syndrome protein (WRN) and model replication forks. The Holliday junction is readily formed once the lagging arm is unwound, and migrated unidirectionally with 3.2 ± 0.03 bases/s velocity. The recovery of the replication fork was controlled by branch migration reversal of WRN, resulting in repetitive fork regression. The Holliday junction formation, branch migration, and migration direction reversal are all ATP dependent, revealing that WRN uses the energy of ATP hydrolysis to actively coordinate the structural transitions of DNA.

Keywords: replication fork regression; single-molecule FRET; werner syndrome helicase.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Intramural

MeSH terms

Adenosine Triphosphate / metabolism*
Animals
Base Pairing
Carbocyanines / chemistry
DNA Replication*
DNA, Cruciform / chemistry*
DNA, Cruciform / genetics
DNA, Cruciform / metabolism
Fluorescence Resonance Energy Transfer
Fluorescent Dyes / chemistry
Gene Expression
Humans
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism*
Sf9 Cells
Single Molecule Imaging
Spodoptera
Werner Syndrome Helicase / genetics
Werner Syndrome Helicase / metabolism*

Substances

Carbocyanines
DNA, Cruciform
Fluorescent Dyes
Recombinant Fusion Proteins
cyanine dye 3
cyanine dye 5
Adenosine Triphosphate
WRN protein, human
Werner Syndrome Helicase

Grants and funding

ZIA AG000721-08/Intramural NIH HHS/United States