In Vitro SUMOylation Assays with a DNA-Sensitive Multi-subunit E3 Ligase

SUMOylation is a dynamic post-translational modification that regulates different cellular processes. The SUMOylation pathway, involving E1, E2, and E3 enzymes, plays essential roles in genome integrity through its participation in numerous chromosomal transactions. The Smc5/6 complex, a member of the Structural Maintenance of Chromosomes (SMC) family, contains a SUMO E3 ligase domain within its Nse2 subunit that mainly targets DNA-associated factors. In vitro experiments using purified SUMO enzymes and Smc5/6 subunits have identified several regulatory elements of this E3, including activation by DNA. Here we present a streamlined protocol for producing SUMO, the SUMO E1 and E2 enzymes, and Smc5/6 complexes as a multi-subunit SUMO E3 ligase. In addition, we describe how these components can be used in vitro to assess regulatory elements of the E3, with a specific focus on the effects of DNA on the activity of the Nse2 SUMO ligase.