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Updated: Jan 8, 2026
Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins
Published on: September 28, 2012
Fmp45 promotes Rad53 dephosphorylation via Ptc2 interaction to attenuate checkpoint signaling and maintain genome
Zhongyi Cong1, Jingyuan Jiang1, Mengyuan Li1
1Department of Regenerative Medicine, School of Pharmaceutical Science, Jilin University, Fujin Road 1266, Changchun 130021, China.
Background:
Double-strand breaks (DSBs) are repaired through the coordinated action of DNA damage checkpoint pathways, homologous recombination (HR), and non-homologous end joining (NHEJ) mechanisms. Given the complexity of DSB repair networks, the identification of novel regulatory factors remains essential for a comprehensive understanding of genomic stability maintenance. Fmp45, a membrane protein previously implicated in salt stress response but with no known role in DNA repair, was found to be upregulated in multiple DNA damage-related transcriptomic datasets (GEO: GSE83454, GSE155701, GSE74642). This observation led us to hypothesize that Fmp45 might represent a previously unrecognized component of the DSB repair machinery. In this study, we demonstrate that Fmp45 functions as a zeocin-specific modulator of the DSB response in Saccharomyces cerevisiae.
Methods:
Growth phenotypes were analysed to investigate FMP45 deletion (fmp45Δ) and its genetic interactions with other genes, protein expression and Rad53 phosphorylation was assessed by Western blotting, protein localization was analysed by Laser confocal microscopy, cell cycle progression was determined by flow cytometry, and protein-protein interactions was probed yeast two-hybrid assays.
Results:
Growth assays revealed that fmp45Δ mutants exhibited hypersensitivity to DSBs induced by zeocin but not to other DNA lesions caused by hydroxyurea, methyl methanesulfonate, 4-nitroquinoline-1-oxide, or camptothecin. Genetic interaction analysis showed that Fmp45 cooperated with checkpoint gene Rad9 but not with HR-related genes (Sae2, Exo1) or NHEJ factor yku70. Laser confocal microscopy confirmed that FMP45 deletion did not impair nuclear localization of Rad51, a key mediator of HR-mediated DNA strand invasion. Further analysis of Rad53 phosphorylation/dephosphorylation dynamics, growth phenotypes of fmp45Δ with phosphatase (ptc2Δ, pph3Δ) and checkpoint effector (rad9Δ, mrc1Δ) mutants, cell cycle profiling, and yeast two-hybrid assays demonstrated that Fmp45 interacts with Ptc2 to promote Rad53 dephosphorylation, thereby preventing excessive cell cycle arrest.
Conclusion:
These findings elucidate a critical checkpoint-phosphatase coordination mechanism ensuring timely DNA damage recovery and genomic stability, identifying Fmp45 as a key regulator balancing DNA repair fidelity with cell cycle resumption following DSBs.
Insights
Fmp45 regulates DNA double-strand break repair by coordinating checkpoint and phosphatase activity. This protein ensures proper cell cycle resumption after DNA damage, maintaining genomic stability.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- DNA double-strand breaks (DSBs) are critical DNA lesions repaired by complex pathways including homologous recombination (HR) and non-homologous end joining (NHEJ).
- Identifying novel regulators of DSB repair is crucial for understanding genomic stability.
- Fmp45, a protein previously linked to salt stress, was found upregulated in DNA damage response datasets, suggesting a potential role in DSB repair.
Purpose of the Study:
- To investigate the role of Fmp45 in DNA double-strand break (DSB) repair in Saccharomyces cerevisiae.
- To determine if Fmp45 is a novel component of the DSB repair machinery.
Main Methods:
- Deletion mutant analysis (fmp45Δ) and genetic interaction studies.
- Western blotting for protein expression and phosphorylation (Rad53).
- Laser confocal microscopy for protein localization, flow cytometry for cell cycle analysis, and yeast two-hybrid assays for protein-protein interactions.
Main Results:
- fmp45Δ mutants showed hypersensitivity to zeocin-induced DSBs but not other DNA damaging agents.
- Fmp45 genetically interacted with the checkpoint gene Rad9 but not HR or NHEJ genes.
- Fmp45 interacts with phosphatase Ptc2 to promote Rad53 dephosphorylation, facilitating cell cycle resumption after DSBs.
Conclusions:
- Fmp45 acts as a zeocin-specific modulator of the DSB response.
- A novel checkpoint-phosphatase coordination mechanism involving Fmp45 ensures timely DNA damage recovery.
- Fmp45 is identified as a key regulator balancing DNA repair fidelity and cell cycle progression post-DSB.
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