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A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells
Published on: March 5, 2018
A novel assay revealed that ribonucleotide reductase is functionally important for interstrand DNA crosslink repair
Naoaki Fujii1, Benjamin J Evison1, Marcelo L Actis1
1Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
Abstract:
Cells have evolved complex biochemical pathways for DNA interstrand crosslink (ICL) removal. Despite the chemotherapeutic importance of ICL repair, there have been few attempts to identify which mechanistic DNA repair inhibitor actually inhibits ICL repair. To identify such compounds, a new and robust ICL repair assay was developed using a novel plasmid that contains synthetic ICLs between a CMV promoter region that drives transcription and a luciferase reporter gene, and an SV40 origin of replication and the large T antigen (LgT) gene that enables self-replication in mammalian cells. In a screen against compounds that are classified as inhibitors of DNA repair or synthesis, the reporter generation was exquisitely sensitive to ribonucleotide reductase (RNR) inhibitors such as gemcitabine and clofarabine, but not to inhibitors of PARP, ATR, ATM, Chk1, and others. The effect was observed also by siRNA downregulation of RNR. Moreover, the reporter generation was also particularly sensitive to 3-AP, a non-nucleoside RNR inhibitor, but not significantly sensitive to DNA replication stressors, suggesting that the involvement of RNR in ICL repair is independent of incorporation of a nucleotide RNR inhibitor into DNA to induce replication stress. The reporter generation from a modified version of the plasmid that lacks the LgT-SV40ori motif was also adversely affected by RNR inhibitors, further indicating a role for RNR in ICL repair that is independent of DNA replication. Intriguingly, unhooking of cisplatin-ICL from nuclear DNA was significantly inhibited by low doses of gemcitabine, suggesting an unidentified functional role for RNR in the process of ICL unhooking. The assay approach could identify other molecules essential for ICLR in quantitative and flexible manner.
Insights
Researchers identified ribonucleotide reductase (RNR) inhibitors as key to DNA interstrand crosslink (ICL) repair. This discovery offers new insights into DNA repair mechanisms and potential therapeutic targets for cancer treatment.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA interstrand crosslinks (ICLs) are DNA lesions that pose a significant challenge to cellular integrity.
- Efficient removal of ICLs is crucial for preventing genomic instability and is a target for cancer chemotherapy.
- Identifying specific inhibitors of ICL repair pathways is essential for understanding these mechanisms and developing targeted therapies.
Purpose of the Study:
- To develop a novel assay for identifying inhibitors of DNA interstrand crosslink (ICL) repair.
- To screen known DNA repair and synthesis inhibitors for their effect on ICL repair.
- To elucidate the specific role of ribonucleotide reductase (RNR) in ICL repair.
Main Methods:
- Development of a novel plasmid-based assay containing synthetic ICLs, a luciferase reporter, and a self-replication system (LgT-SV40ori) in mammalian cells.
- Screening of various DNA repair and synthesis inhibitors, including those targeting PARP, ATR, ATM, Chk1, and ribonucleotide reductase (RNR).
- Utilizing siRNA to downregulate RNR expression and assessing the impact on reporter gene expression.
- Testing a modified plasmid lacking the replication origin to confirm RNR's role independent of DNA replication.
Main Results:
- The developed ICL repair assay was highly sensitive to ribonucleotide reductase (RNR) inhibitors, such as gemcitabine and clofarabine, but not to inhibitors of other DNA repair pathways (PARP, ATR, ATM, Chk1).
- siRNA-mediated knockdown of RNR confirmed its essential role in ICL repair.
- RNR inhibition affected reporter generation independently of DNA replication stress, indicating a distinct role in ICL repair.
- Low doses of gemcitabine significantly inhibited the unhooking of cisplatin-induced ICLs, suggesting a functional role for RNR in this process.
Conclusions:
- Ribonucleotide reductase (RNR) plays a critical, previously unrecognized role in DNA interstrand crosslink (ICL) repair, particularly in the unhooking step.
- The novel assay is a robust and flexible tool for identifying molecules involved in ICL repair.
- Targeting RNR could represent a novel strategy for enhancing the efficacy of ICL-inducing chemotherapies.
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