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Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
Published on: August 21, 2021
DNA maintenance following bleomycin-induced strand breaks does not require poly(ADP-ribosyl)ation activation in
Layal Ishak1, Amandine Moretton1, Isabelle Garreau-Balandier1
1Université Clermont Auvergne, Université Blaise Pascal, EA 4645, Réparation du Génome Mitochondrial Normal et Pathologique, BP 10448, F-63000 Clermont-Ferrand, France.
Background:
Poly-ADP ribosylation (PARylation) is a post translational modification, catalyzed by Poly(ADP-ribose)polymerase (PARP) family. In Drosophila, PARP-I (human PARP-1 ortholog) is considered to be the only enzymatically active isoform. PARylation is involved in various cellular processes such as DNA repair in case of base excision and strand-breaks.
Observations:
Strand-breaks (SSB and DSB) are detrimental to cell viability and, in Drosophila, that has a unique PARP family organization, little is known on PARP involvement in the control of strand-breaks repair process. In our study, strands-breaks (SSB and DSB) are chemically induced in S2 Drosophila cells using bleomycin. These breaks are efficiently repaired in S2 cells. During the bleomycin treatment, changes in PARylation levels are only detectable in a few cells, and an increase in PARP-I and PARP-II mRNAs is only observed during the recovery period. These results differ strongly from those obtained with Human cells, where PARylation is strongly activating when DNA breaks are generated. Finally, in PARP knock-down cells, DNA stability is altered but no change in strand-breaks repair can be observed.
Conclusions:
PARP responses in DNA strands-breaks context are functional in Drosophila model as demonstrated by PARP-I and PARP-II mRNA increases. However, no modification of the global PARylation profile is observed during strand-breaks generation, only changes at cellular levels are detectable. Taking together, these results demonstrate that PARylation process in Drosophila is tightly regulated in the context of strands-breaks repair and that PARP is essential during the maintenance of DNA integrity but dispensable in the DNA repair process.
Insights
Poly-ADP ribosylation (PARylation) in Drosophila is tightly regulated during DNA strand-break repair. While Poly(ADP-ribose)polymerase (PARP) is crucial for DNA integrity, it appears dispensable for the repair process itself.
Area of Science:
- Molecular Biology
- Genetics
- Cellular Biology
Background:
- Poly-ADP ribosylation (PARylation) is a critical post-translational modification catalyzed by the Poly(ADP-ribose)polymerase (PARP) family.
- In Drosophila, PARP-I is the primary active isoform, involved in cellular processes like DNA repair.
- PARylation plays a role in repairing DNA base excision and strand breaks.
Purpose of the Study:
- Investigate the role of PARP in controlling DNA strand-break repair in Drosophila.
- Compare PARP involvement in DNA repair between Drosophila and human cells.
- Determine the necessity of PARP in maintaining DNA integrity and repair.
Main Methods:
- Chemically induced single-strand breaks (SSB) and double-strand breaks (DSB) in S2 Drosophila cells using bleomycin.
- Monitored PARylation levels and PARP-I/PARP-II mRNA expression.
- Assessed DNA stability and strand-break repair efficiency in PARP knock-down cells.
Main Results:
- Drosophila S2 cells efficiently repaired induced DNA strand breaks.
- PARylation levels showed minimal changes during bleomycin treatment, with increases in PARP-I and PARP-II mRNA observed post-treatment.
- PARP knock-down altered DNA stability but did not impede strand-break repair.
- Observed differences in PARP response compared to human cells where PARylation is strongly activated by DNA breaks.
Conclusions:
- PARP-mediated DNA strand-break repair responses are functional in Drosophila, evidenced by increased PARP mRNA.
- Global PARylation profiles remain largely unchanged during DNA break generation in Drosophila.
- PARylation is tightly regulated in Drosophila during DNA strand-break repair, with PARP essential for DNA integrity but not the repair process itself.
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