Role of Nse1 Subunit of SMC5/6 Complex as a Ubiquitin Ligase
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plasmids
2.2. Expression and Purification of Recombinant Proteins
2.3. In Vitro Ubiquitination Assay
2.4. Yeast Two-Hybrid Assay
2.5. Yeast Strains
2.6. Tandem Affinity Purification of Nse1-HBH
2.7. Liquid Chromatography-Mass Spectrometry Analyses
2.8. Protein Modelling
3. Results
3.1. Nse1 Is a Ubiquitin Ligase
3.2. Nse1 Ligase Activity Is Abolished by the Nse1-R188E Mutation
3.3. Nse1 Associates with Ubc13/Mms2 and Ubiquitin
3.4. Nse1 E3 Activity Plays an Important Role during Replication Stress
3.5. Nse4 and Nse3 Are Substrates of Nse1 Ubiquitin Ligase
4. Discussion
4.1. Nse1 Is a Ubiquitin Ligase Working Specifically with Ubc13/Mms2
4.2. Nse1 Mutation R188E Specifically Impairs Its Ubiquitin Ligase Function
4.3. Nse1 Ligase Substrates
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Kolesar, P.; Stejskal, K.; Potesil, D.; Murray, J.M.; Palecek, J.J. Role of Nse1 Subunit of SMC5/6 Complex as a Ubiquitin Ligase. Cells 2022, 11, 165. https://doi.org/10.3390/cells11010165
Kolesar P, Stejskal K, Potesil D, Murray JM, Palecek JJ. Role of Nse1 Subunit of SMC5/6 Complex as a Ubiquitin Ligase. Cells. 2022; 11(1):165. https://doi.org/10.3390/cells11010165
Chicago/Turabian StyleKolesar, Peter, Karel Stejskal, David Potesil, Johanne M. Murray, and Jan J. Palecek. 2022. "Role of Nse1 Subunit of SMC5/6 Complex as a Ubiquitin Ligase" Cells 11, no. 1: 165. https://doi.org/10.3390/cells11010165
APA StyleKolesar, P., Stejskal, K., Potesil, D., Murray, J. M., & Palecek, J. J. (2022). Role of Nse1 Subunit of SMC5/6 Complex as a Ubiquitin Ligase. Cells, 11(1), 165. https://doi.org/10.3390/cells11010165