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- Title
Detection the specificity of poly(ADP-ribose) polymerase 1 and poly(ADP-ribose) polymerase-2 interaction with DNA strand breaks at single molecule level.
- Authors
Sukhanova, Maria V.; Abrakhi, Sanae; Joshi, Vandana; Anabaev, Rashid O.; Kutuzov, Mikhail M.; Pastré, David; Curmi, Patrick; Hamon, Loic; Lavrik, Olga I.
- Abstract
The aim of this study was to investigate the interaction of PARP1 and PARP2 with single DNA damage site in the context of long DNA substrates. Methods: The interaction PARP1 and PARP2 with long DNA fragments containing DNA breaks was studied using atomic force microscopy (AFM) and fluorescent titration. Results: PARP1 and PARP2 are eukaryotic nuclear proteins which are implicated in synthesis of poly(ADP-ribose) (PAR) after DNA damage detection. We directly visualized and compared the binding of PARP1 and PARP2 to long DNA fragments (1200-bp) containing only double-strand breaks (DSBs) or DSB along with a unique nick (nicked 1200-bp). AFM data show that PARP1 specifically binds to both nick and DSBs. At the same time, this protein binds to undamaged DNA, but to a lower level than to breaks. In contrast to PARP1, PARP2 binds weakly to undamaged DNA and to DSBs, and localizes mainly to nick site. These results correlate with fluorescence measurement data revealing that the binding affinity of PARP2 to 1200 dsDNA is 5-fold weaker in comparison to the same DNA fragment containing a nick. Interestingly, AFM assay clear demonstrate that PARylated PARP1 and PARP2 are still able to interact with DSB and nick. Conclusion: Our AFM observations at single-molecule level implicate PARP2 in the recognition of nick and suggest its role in single-strand break repair. It is appears that the length of the PAR polymer formed by PARP1 and PARP2 proteins is influenced by the initial binding affinity of the proteins for the damaged DNA site.
- Subjects
POLY(ADP-ribose) polymerase; ATOMIC force microscopy; SINGLE-strand DNA breaks
- Publication
Biopolymers & Cell, 2015, p30
- ISSN
0233-7657
- Publication type
Article