Updated project metadata. Structural Maintenance of Chromosomes (SMC) complexes - cohesin, condensin and SMC5/6 - are important for many aspects of the chromosomal organization. These complexes are composed of Smc and non-Smc element (Nse) subunits. The Nse1 subunit of SMC5/6 contains a variant RING domain, characteristic of ubiquitin ligases. Human NSE1 has been shown to possess ubiquitin ligase activity in vitro; however, other studies were unable to show such activity. Here, we confirm Nse1 ubiquitin-ligase activity in vitro using purified Schizosaccharomyces pombe proteins. We demonstrate that the Nse1 ligase activity is stimulated by Nse3 and Nse4, it specifically utilizes Ubc13/Mms2 E2 enzyme and Nse1 interacts directly with ubiquitin. We identify Nse1 mutation (R188E) that specifically disrupts its E3 activity, and show that the Nse1-dependent ubiquitination is particularly important under replication stress. Moreover, we identify Nse4 (lysine K181) as the first known SMC5/6-associated Nse1 substrate. Interestingly, abolition of Nse4 modification at K181 leads to suppression of DNA-damage sensitivity of other SMC5/6 mutants. Altogether, this study brings new evidence for Nse1 ubiquitin ligase activity, significantly advancing our under-standing of this enigmatic SMC5/6 function.