Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
DNA Repair (Amst). 2012 Oct 1;11(10):799-810. doi: 10.1016/j.dnarep.2012.06.010. Epub 2012 Aug 24.
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.
Nse2/Mms21 是 SMC5/6 复合物的 E3 SUMO 连接酶组件，在维持基因组稳定性方面发挥多种作用。为了研究脊椎动物 Nse2 同源物的功能，我们生成了 Nse2 缺陷型鸡 DT40 细胞。Nse2 对于 DT40 细胞的活力并非必需，但对于大体积 DNA 损伤的有效修复是必需的，尽管 Nse2 缺陷型细胞对电离辐射诱导的 DNA 损伤表现出正常的敏感性。同源重组活性在 Nse2(-/-/-)细胞中降低。Nse2 缺陷使 Smc5 不稳定，但 Smc6 不受影响。在挽救实验中，我们发现 Nse2 的 SUMO 连接酶活性对于有效应对 MMS 或顺铂诱导的 DNA 损伤以及同源重组是必需的，但对于 Smc5 的稳定性并非必需。凝胶过滤分析表明，Smc5 和 Nse2 在细胞周期内和 DNA 损伤后仍然保持关联，并且 Smc5/Smc6 关联不依赖于 Nse2。对虽然生长缓慢但仍存活的 Nse2(-/-/-)Smc5(-)克隆进行分析，未发现 DNA 损伤敏感性显著增加。我们提出，Nse2 决定了脊椎动物细胞中 Smc5/6 复合物的活性，但不决定其组装，而这种活性需要 Nse2 SUMO 连接酶功能。
