Abstract
Genomic stability is maintained by the concerted actions of numerous protein complexes that participate in chromosomal duplication, repair, and segregation. The Smc5/6 complex is an essential multi-subunit complex crucial for repair of DNA double-strand breaks. Two of its subunits, Nse1 and Nse3, are homologous to the RING-MAGE complexes recently described in human cells. We investigated the contribution of the budding yeast Nse1 RING-domain by isolating a mutant nse1-103 bearing substitutions in conserved Zinc-coordinating residues of the RING-domain that is hypersensitive to genotoxic stress and temperature. The nse1-103 mutant protein was defective in interaction with Nse3 and other Smc5/6 complex subunits, Nse4 and Smc5. Chromosome loss was enhanced, accompanied by a delay in the completion of replication and a modest defect in sister chromatid cohesion, in nse1-103. The nse1-103 mutant was synthetic sick with rrm3∆ (defective in fork passage through pause sites), this defect was rescued by inactivation of Tof1, a subunit of the fork protection complex that enforces pausing. The temperature sensitivity of nse1-103 was partially suppressed by deletion of MPH1, encoding a DNA-helicase. Homology modeling of the structure of the budding yeast Nse1–Nse3 heterodimer based on the human Nse1–MAGEG1 structure suggests a similar organization and indicates that perturbation of the Zn-coordinating cluster has the potential to allosterically alter structural elements at the Nse1/Nse3 interaction interface that may abrogate their association. Our findings demonstrate that the budding yeast Nse1 RING-domain organization is important for interaction with Nse3, which is crucial for completion of chromosomal replication, cohesion, and maintenance of chromosome stability.
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(adapted from Huang and Koshland 2003; Huang and Koshland 2003). b Chromosome instability in nse1-103 cells grown at 23 °C. Quantification of telomere marker loss (YAC integrity; top panels) and total YAC loss (YAC stability; bottom panels) plotted as percent loss on the y-axis in WT (OR SLY2583 and SLY2571) and nse1-103 (SLY2584 and SLY2572) cells that harbored the YACs PA3-I (left panels) and yWSS1572-1 (right panels) are shown. Error bars indicate standard error of the mean. The unpaired two-tailed Student’s t test was used to evaluate the statistical significance; double asterisk indicates a statistically significant difference and implies a P value that is < 0.01 (P = 0.0047 in this case)
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Abbreviations
- Smc:
-
Structural maintenance of chromosomes
- MMS:
-
Methyl methane sulfonate
- HU:
-
Hydroxyurea
- Mms21:
-
MMS-sensitive 21
- SUMO:
-
Small ubiquitin-related modifier
- Nse:
-
Non-smc element
- DSB:
-
Double-strand break
- GCR:
-
Gross chromosomal rearrangements
- GST:
-
Glutathione S-transferase
- MAGEG1:
-
Melanoma antigen G1
- MPH1:
-
Mutator Phenotype
- PFGE:
-
Pulsed-field gel electrophoresis
- Rad:
-
Radiation-sensitive
- rDNA:
-
Ribosomal DNA
- RING:
-
Really interesting new gene
- Rrm3:
-
rDNA recombination mutation 3
- Tof1:
-
Topoisomerase I-interacting Factor 1
- YPD:
-
Yeast extract–peptone–dextrose
- SC:
-
Synthetic complete medium
- YAC:
-
Yeast artificial chromosome
- 5-FOA:
-
5-fluoroorotic acid
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Acknowledgements
This work was supported by investigator specific grants to Shikha Laloraya from the Science and Engineering Research Board (SERB), Department of Biotechnology (DBT) and Council of Scientific and Industrial Research (CSIR), India, and the DBT-IISc partnership program funded by the Department of Biotechnology, India. Fellowship support for Saima Wani was provided by the Indian Institute of Science and the SERB grant to S.L. Deepash Kothiwal was supported by a Department of Biotechnology Senior Research Fellowship, Lakshmi Mahendrawada by the CSIR grant to S.L., and Neelam Maharshi by a fellowship from I.I.Sc. We thank Yves Barral, Kenji Kohno and Doug Koshland for sharing strains and plasmids, Donald Soubam for construction of pDS58 and pDS72, Deepa Balagopal for construction of pDB30, and N. Srinivasan for discussions related to the structure. Technical support from the DBT funded confocal facility of I.I.Sc. is acknowledged. Equipment support for the Department of Biochemistry, I.I.Sc. is provided by the DST-FIST and UGC CAS/SAP programs.
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S.M. and S.L. designed the experiments. S.M., N.M., L.M. and D.K. performed the experiments. S.M., N.M., D.K., L.M. and S.L. analyzed the data. S.L. performed the homology modeling and K.R. analyzed and interpreted the Nse1/3 heterodimer model. S.L. designed and supervised the project and wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript.
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Communicated by M. Kupiec.
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Wani, S., Maharshi, N., Kothiwal, D. et al. Interaction of the Saccharomyces cerevisiae RING-domain protein Nse1 with Nse3 and the Smc5/6 complex is required for chromosome replication and stability. Curr Genet 64, 599–617 (2018). https://doi.org/10.1007/s00294-017-0776-6
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DOI: https://doi.org/10.1007/s00294-017-0776-6