New insights into the role of condensin in Saccharomyces cerevisiae
Structural Maintenance of Chromosomes (SMC) complexes are highly conserved from prokaryotes to eukaryotes. Saccharomyces cerevisiae has six SMC proteins which form three SMC-complexes in association with non-SMC subunits namely, the cohesin complex, the condensin complex, and the Smc5/6 complex. The condensin complex plays an essential role in chromosome condensation and is also involved in various other chromosomal processes such as sister chromatid cohesion, transcriptional silencing, rDNA segregation, tRNA-gene clustering etc. Condensin subunits undergo post-translational modifications such as phosphorylation and sumoylation. In order to understand the role of sumoylation of condensin, we created a sumoylation-defective condensin mutant that shows an increased sensitivity towards DNA damaging agents such hydroxyurea (HU), methyl methane sulfonate (MMS) and ultraviolet (UV) radiation. We observed accumulation of Ddc2 foci in the mutant suggesting the presence of DNA damage. To understand the role of sumoylation of condensin in genomic stability, we performed a YAC based genetic assay for chromosome stability. We observed a significant enhancement in chromosome loss frequency in the mutant compared to wild-type cells. This is accompanied by a modest reduction in sister chromatid arm cohesion in the mutant, that may be a potential reason for the observed chromosome instability. Since condensin is known to be involved in transcriptional silencing, we performed silencing assays at all three known silenced loci in the mutant cells, to explore the effect of sumoylation of condensin on gene expression. We found a reduction in the extent of silencing of the silencing reporter at telomeres in the strain expressing sumoylation-defective condensin whereas silencing was enhanced at the rDNA locus. However, silencing at the mating-type loci was unaltered. We found that the expression of native telomere proximal genes was also increased in the mutant. The histone deacetylase, Sir2, that is required for silencing, is redistributed from telomeres to the rDNA in the mutant. Although condensin brings about condensation of chromosomes during mitosis (M-phase), it binds to chromatin throughout the cell cycle. Condensin’s potential functions in the maintenance of genomic integrity during interphase remain elusive. We observed that one of the condensin mutants showed a severe sensitivity to hydroxyurea, a replication inhibitor, and exhibits activation of the intra S-phase checkpoint in absence of extrinsic genotoxic stress. To further explore its role in S-phase, we created a cell cycle regulated allele of condensin that limits its expression to the G2/M phase. Restricting the levels of condensin to the G2/M phase did not affect cell survival, indicating its essential role in mitosis. Interestingly, these cells that lack condensin during G1/S phase showed enhanced sensitivity to hydroxyurea. The mutant showed a delay in cell cycle progression particularly in increasing the DNA content from 1C to 2C. Replication kinetics was delayed in the absence of condensin during S-phase. Furthermore, we observed that the binding of a replication fork component to replication origins was prolonged, suggesting a delay in the progression of replication forks. Genetic interaction analysis revealed that the proteins involved in intra-S phase checkpoint (but not DNA damage checkpoint), replication fork restart pathway and homologous recombination pathway, are required for growth of the condensin mutant, both in presence and absence of extrinsic DNA-replication stress. In conclusion, our present study emphasizes the functional significance of sumoylation of the condensin complex in maintaining genomic stability. Also, condensin sumoylation is important for sub-telomeric silencing and in regulating the distribution of the limited pool of Sir2 between the nucleolus and extra-nucleolar regions within the nucleus. In addition, condensin is important for S-phase progression and timely completion of replication.
- Biochemistry (BC)