Interaction of the Saccharomyces cerevisiae RING-domain protein Nse1 with Nse3 and the Smc5/6 complex is required for chromosome replication and stability

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.

SMC complexes organize the bacterial chromosome by lengthwise compaction

“…The models, which also assume that MukBEF loads randomly on all chromosomal regions, explain how MukBEF clusters colocalize with the replication origin region (oriC) in wild-type cells, while MukBEF clusters localize equally with all genetic regions tested in MatP − cells. (Kakui and Uhlmann 2018;Wani et al 2018;Paul et al 2019;Yatskevich et al 2019) Is the MukBEF-organized E. coli chromosome placed randomly within a cell? Early imaging studies showed that in new-born E. coli cells that have not initiated replication, the left and right replichores are organized into separate cell halves, while oriC is at midcell (Wang et al 2006).…”

“…Distant relations to the SMC complexes above are Rad50 and RecN, involved in repair of double-strand DNA breaks (not shown). For further details see (Kakui and Uhlmann 2018 ; Wani et al 2018 ; Paul et al 2019 ; Yatskevich et al 2019 ) …”

SMC5/6: Multifunctional Player in Replication

“…Another piece of data also points to natural RPSs, as SMC5/6 is enriched at RPSs (rDNA, tDNA, centromeres, and telomeres) and co-localizes with Rrm3 helicase, which facilitates fork passage through these sites [84,112,113]. The sickness/lethality of the smc6 rrm3∆ double mutant is rescued by individual deletions of Tof1-Csm3 (a fork protection complex that enforces pausing at RPS).…”

Smc5/6, an atypical SMC complex with two RING-type subunits

“…Interestingly, mutations in zinc-coordinating residues in fission yeast suppress the repair defects of hypomorphic Smc5/6 mutants, probably by preventing the recruitment of dysfunctional complexes to damaged loci [114]. In budding yeast, NH-RING mutants show delayed DNA replication, modest sister chromatid cohesion defects and increased spontaneous chromosome loss events [115]. Interestingly, the NH-RING in Nse1 has been related to two disparately different functions (Figure 2A).…”

“…Interestingly, the NH-RING in Nse1 has been related to two disparately different functions (Figure 2A). On one hand, the yeast NH-RING of Nse1 is necessary for the stability of the Nse1-Nse3-Nse4 subcomplex, and mutations in the NH-RING disrupt the Nse1-Nse4 interaction, resulting in Nse1-Nse3 dimers with defective binding to Nse4 [30,115]. On the other hand, the human NH-RING displays a weak E3 ubiquitin ligase activity in vitro that is greatly stimulated in the presence of Nse3 [32].…”