Ii Tatsuya, Mullen Janet R, Slagle Christopher E, Brill Steven J
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA.
DNA Repair (Amst). 2007 Nov;6(11):1679-91. doi: 10.1016/j.dnarep.2007.06.004. Epub 2007 Jul 31.
The yeast genes SLX5 and SLX8 were identified based on their requirement for viability in the absence of the Sgs1 DNA helicase. Loss of these genes results in genome instability, nibbled colonies, and other phenotypes associated with defects in sumoylation. The Slx5 and Slx8 proteins form a stable complex and each subunit contains a single RING-finger domain at its C-terminus. To determine the physiological function of the Slx5-8 complex, we explored its interaction with the SUMO pathway. Curing 2micro circle from the mutants, suppressed their nibbled colony phenotype and partially improved their growth rate, but did not affect their sensitivity to hydroxyurea. The increase in sumoylation observed in slx5Delta and slx8Delta mutants was found to be dependent on the Siz1 SUMO ligase. Physical interactions between the Slx5-8 complex and both Ubc9 and Smt3 were identified and characterized. Using in vitro reactions, we show that Slx5, Slx8, or the Slx5-8 complex stimulates the formation of SUMO chains and the sumoylation of a test substrate. Interestingly, a functional RING-finger domain is not required for this stimulation in vitro. These biochemical data demonstrate for the first time that the Slx5 and Slx8 complex is capable of interacting directly with the SUMO pathway.
酵母基因SLX5和SLX8是根据它们在缺乏Sgs1 DNA解旋酶时对生存力的需求而被鉴定出来的。这些基因的缺失会导致基因组不稳定、边缘参差不齐的菌落以及其他与SUMO化缺陷相关的表型。Slx5和Slx8蛋白形成一个稳定的复合物，并且每个亚基在其C端都含有一个单一的泛素连接酶结构域。为了确定Slx5-8复合物的生理功能，我们探究了它与SUMO途径的相互作用。从突变体中去除2微米质粒，抑制了它们边缘参差不齐的菌落表型，并部分提高了它们的生长速率，但不影响它们对羟基脲的敏感性。在slx5Δ和slx8Δ突变体中观察到的SUMO化增加被发现依赖于Siz1 SUMO连接酶。鉴定并表征了Slx5-8复合物与Ubc9和Smt3之间的物理相互作用。通过体外反应，我们表明Slx5、Slx8或Slx5-8复合物刺激了SUMO链的形成以及一个测试底物的SUMO化。有趣的是，在体外这种刺激不需要一个功能性的泛素连接酶结构域。这些生化数据首次证明Slx5和Slx8复合物能够直接与SUMO途径相互作用。
