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

Saima Wani, Neelam Maharshi, Deepash Kothiwal, Lakshmi Mahendrawada, Raju Kalaivani, Shikha Laloraya

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.