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Single-molecule force spectroscopy reveals binding and bridging dynamics of PARP1 and PARP2 at DNA double-strand breaks
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Bell, Nicholas A. W. and Molloy, Justin E. (2023) Single-molecule force spectroscopy reveals binding and bridging dynamics of PARP1 and PARP2 at DNA double-strand breaks. Proceedings of the National Academy of Sciences, 120 (22). e2214209120. doi:10.1073/pnas.2214209120 ISSN 0027-8424.
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WRAP-single-molecule-force-spectroscopy-reveals-binding-bridging-dynamics-PARP1-PARP2-DNA-double-strand-breaks-Molloy-2023.pdf - Published Version - Requires a PDF viewer. Available under License Creative Commons Attribution 4.0. Download (2274Kb) | Preview |
Official URL: https://doi.org/10.1073/pnas.2214209120
Abstract
Poly(ADP-ribose) polymerases (PARPs) play key roles in DNA damage repair pathways in eukaryotic cells. Human PARPs 1 and 2 are catalytically activated by damage in the form of both double-strand and single-strand DNA breaks. Recent structural work indicates that PARP2 can also bridge two DNA double-strand breaks (DSBs), revealing a potential role in stabilizing broken DNA ends. In this paper, we have developed a magnetic tweezers–based assay in order to measure the mechanical stability and interaction kinetics of proteins bridging across the two ends of a DNA DSB. We find that PARP2 forms a remarkably stable mechanical link (rupture force ~85 pN) across blunt-end 5′-phosphorylated DSBs and restores torsional continuity allowing DNA supercoiling. We characterize the rupture force for different overhang types and show that PARP2 switches between bridging and end-binding modes depending on whether the break is blunt-ended or has a short 5′ or 3′ overhang. In contrast, PARP1 was not observed to form a bridging interaction across blunt or short overhang DSBs and competed away PARP2 bridge formation, indicating that it binds stably but without linking together the two broken DNA ends. Our work gives insights into the fundamental mechanisms of PARP1 and PARP2 interactions at double-strand DNA breaks and presents a unique experimental approach to studying DNA DSB repair pathways.
Item Type: | Journal Article | |||||||||||||||
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Subjects: | Q Science > QH Natural history > QH426 Genetics Q Science > QP Physiology |
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Divisions: | Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School | |||||||||||||||
SWORD Depositor: | Library Publications Router | |||||||||||||||
Library of Congress Subject Headings (LCSH): | DNA repair, DNA damage, DNA-protein interactions, Eukaryotic cells, NAD-ADP-ribosyltransferase, Glycosyltransferases | |||||||||||||||
Journal or Publication Title: | Proceedings of the National Academy of Sciences | |||||||||||||||
Publisher: | National Academy of Sciences | |||||||||||||||
ISSN: | 0027-8424 | |||||||||||||||
Official Date: | 22 May 2023 | |||||||||||||||
Dates: |
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Volume: | 120 | |||||||||||||||
Number: | 22 | |||||||||||||||
Article Number: | e2214209120 | |||||||||||||||
DOI: | 10.1073/pnas.2214209120 | |||||||||||||||
Status: | Peer Reviewed | |||||||||||||||
Publication Status: | Published | |||||||||||||||
Access rights to Published version: | Open Access (Creative Commons) | |||||||||||||||
Date of first compliant deposit: | 18 July 2023 | |||||||||||||||
Date of first compliant Open Access: | 19 July 2023 | |||||||||||||||
RIOXX Funder/Project Grant: |
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