Abstract
Nse2/Mms21 is an E3 SUMO ligase component of the Smc5/6 complex, which plays multiple roles in maintaining genome stability. To study the functions of the vertebrate Nse2 orthologue, we generated Nse2-deficient chicken DT40 cells. Nse2 was dispensable for DT40 cell viability and required for efficient repair of bulky DNA lesions, although Nse2-deficient cells showed normal sensitivity to ionising radiation-induced DNA damage. Homologous recombination activities were reduced in Nse2(-/-/-) cells. Nse2 deficiency destabilised Smc5, but not Smc6. In rescue experiments, we found that the SUMO ligase activity of Nse2 was required for an efficient response to MMS- or cis-platin-induced DNA damage, and for homologous recombination, but not for Smc5 stability. Gel filtration analysis indicated that Smc5 and Nse2 remain associated during the cell cycle and after DNA damage and Smc5/Smc6 association is independent of Nse2. Analysis of Nse2(-/-/-)Smc5(-) clones, which were viable although slow-growing, showed no significant increase in DNA damage sensitivity. We propose that Nse2 determines the activity, but not the assembly, of the Smc5/6 complex in vertebrate cells, and this activity requires the Nse2 SUMO ligase function.