Single-molecule force spectroscopy reveals binding and bridging dynamics of PARP1 and PARP2 at DNA double-strand breaks

• DOI: 10.1073/pnas.2214209120
• Publication Date: 2023-05-22T19:02:10Z
• Authors: Nicholas A. W. Bell; Justin E. Molloy
• Keywords: Force Spectroscopy; Magnetic tweezers; PARP1

Poly(ADP-ribose) polymerases (PARPs) play key roles in DNA damage repair pathways eukaryotic cells. Human PARPs 1 and 2 are catalytically activated by the form of both double-strand single-strand breaks. Recent structural work indicates that PARP2 can also bridge two breaks (DSBs), revealing a potential role stabilizing broken ends. In this paper, we have developed magnetic tweezers–based assay order to measure mechanical stability interaction kinetics proteins bridging across ends DSB. We find forms remarkably stable link (rupture force ~85 pN) blunt-end 5′-phosphorylated DSBs restores torsional continuity allowing supercoiling. characterize rupture for different overhang types show switches between end-binding modes depending on whether break is blunt-ended or has short 5′ 3′ overhang. contrast, PARP1 was not observed blunt competed away formation, indicating it binds stably but without linking together Our gives insights into fundamental mechanisms interactions at presents unique experimental approach studying DSB pathways.