Molecular analysis of SMC5/6-dependent sumoylation and ubiquitination

Resumen

Eukaryotic cells devote large efforts to maintain the integrity of their genome. The Structural Maintenance of Chromosomes (SMC) complexes, which include cohesin, condensin and Smc5/6, coordinate multiple chromosomal activities that safeguard our genome. Particularly, the Smc5/6 complex plays crucial roles in DNA repair by homologous recombination, replication fork stability and sister chromatid resolution, and it is the most unknown member of the SMC family. Unlike cohesin and condensin, it contains two RING-type domains: one in the Nse1 subunit, with potential ubiquitin ligase activity, and the other in the Nse2 subunit, which has been shown to mediate the transfer of SUMO to substrate proteins. Nse2 binds to the Smc5 coiled-coil through its essential N-terminal domain, whereas its C-terminal half, coding for the SUMO ligase domain, is dispensable for cell survival. Despite this, Nse2-dependent sumoylation of SMC complexes and other chromosomal targets has been reported to control several biological pathways directly involved in genome integrity. However, the processes that regulate its E3 ligase activity remain poorly understood. In this study, we describe a novel mechanism by which the interaction between a positively-charged patch in the coiled-coil of Smc5 and DNA stimulates the Nse2 SUMO E3 ligase activity. In addition, we have performed a detailed functional analysis of the different structural features present in the C-terminal domain of Nse2 in yeast. This characterization reveals that the last C-terminal alpha-helix, which has been related to a rare genetic disorder, has an important structural function and directly affects Nse2 stability. In addition, we have identified two regions that enhance sumoylation in vitro. To our surprise, our results also show that mutations in conserved residues coordinating the zinc ion do not impair Smc5-sumoylation in vivo. The other RING-type subunit of the Smc5/6 complex, Nse1, has also been described to promote DNA repair functions and to maintain genome stability. However, no targets for its ubiquitin-E3 ligase activity have been identified until now. Here, we use label-free quantitative proteomics to compare the ubiquitinome of wild type and nse1 RING mutant cells. Particularly, the largest subunit of the RNA POL I, Rpa190, is less ubiquitinated in nse1 mutant cells. Rpa190 is modified during active transcription, and non-ubiquitinable rpa190-KR cells are sensitive to transcriptional elongation inhibitors and are resistant to BMH-21-mediated proteasomal degradation. Overall, these results provide novel information on the regulation and targets of the Smc5/6-dependent SUMO and ubiquitin ligase activities, which are a critical part of the mechanisms used by the Smc5/6 complex to preserve the integrity of the genome.