Structural Maintenance of Chromosomes (SMC) proteins are a highly conserved class of proteins required for the maintenance of genome stability and regulate nearly all aspects of chromosome biology. Eukaryotes, such as the budding yeast Saccharomyces cerevisiae, have six Smc proteins that form three SMC complexes in association with non-SMC proteins, i.e., the cohesin complex, the condensin complex and the Smc5/6 complex. The yeast Smc5/6 complex consists of Smc5, Smc6 and six non-Smc elements (Nse1-6) that are all essential for the survival of cells.
Nse1 is the first non-smcelement that was identified associated with the Smc5/6 complex. Nse1 has a C-terminal RING-domain, which is a characteristic feature of some E3 ubiquitin ligases. A RING domain consists of eight conserved Zn-coordinating residues arranged in a cross-brace conformation. To understand the importance of this domain, we created site directed mutations in conserved residues identified by sequence alignment of the budding yeast Nse1 RING domain with that of other species. We found a new RING domain mutant nse1-103that was temperature sensitive at 37°C and showed an increased sensitivity towards genotoxic agents such as hydroxyurea (HU), methyl methane sulfonate (MMS) and ultraviolet (UV) radiation. Thense1-103 mutant cells are slow growing and show delayed chromosomal replication at the restrictive temperature. Genetic interactions with replication factors such as RRM3, TOF1 etc. revealed thatnse1-103shows a synthetic sick growth defect in combination with rrm3∆ that is partially suppressed by deletion of TOF1. We found an enhancement in chromosome loss in nse1-103 compared to wild type cells. This was accompanied by a slight reduction in cohesion between the sister chromatids in nse1-103,suggesting a plausible mechanism for the chromosome destabilization observed in the mutant.
Since Nse1 forms part of a trimeric sub-complex with Nse3 and Nse4 in the Smc5/6 complex, we performed a yeast two hybrid assay to test the interaction of nse1-103 with Nse3 or Nse4, and found a defect in interaction of nse1-103 with Nse3 and Nse4. In addition, a defect in association of nse1-103 with Smc5 or Smc6 could be observed by performing co-immunoprecipitation from yeast cell lysates, suggesting that the integrity of the RING-domain is critical for the interaction of Nse1 with other subunits of the Smc5/6 complex. However, there was no defect in the interaction between Nse3 and Smc5 in nse1-103, indicating that the interaction of these components within the complex isindependent of Nse1.
We also identified a novel sequence motif near the RING domain of Nse1, deletion of which leads to an increased sensitivity towards genotoxic stressors and higher temperature. Biochemical characterization of this mutant also suggests a defect ininteraction with Nse3 or Nse4, and also with Smc5. The nse1 mutants also showed defects in post translational modification of Smc5 and other proteins.
Since the Smc5/6 complex also has a SUMO E3 ligase, Mms21/Nse2, we also investigated genetic interactions between the RING domain mutant,nse1-103 and the SUMO ligase RING domain defective mutant,mms21∆sl, and found an exacerbation of the drug sensitive phenotypes in thense1-103 mms21∆sl double mutant relative to either of the single mutants nse1-103 or mms21∆sl, indicating that the two proteins contribute independently to the function of Smc5/6 complex in resisting genotoxic stress.
In conclusion, the present study emphasizes the role of the RING domain of budding yeast Nse1 in resisting genotoxic stress and maintaining chromosome stability and reveals that the integrity of the RING-domain is critical for interactions of Nse1 with Nse3 and other Smc5/6 complex components. In addition, we report identification of another novel sequence motif in Nse1 that is also crucial for its interaction with other subunits of the Smc5/6 complex and for maintenance of post-translational modifications of some cellular proteins.
| Identifer | oai:union.ndltd.org:IISc/oai:etd.iisc.ernet.in:2005/3570 |
| Date | January 2017 |
| Creators | Wani, Saima Masood |
| Contributors | Shikha, Laloraya |
| Source Sets | India Institute of Science |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Relation | G28422 |
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