Department of Biochemistry, Microbiology & Immunology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
PLoS One. 2024 Jul 25;19(7):e0307590. doi: 10.1371/journal.pone.0307590. eCollection 2024.
Telomere resolvases are a family of DNA cleavage and rejoining enzymes that produce linear DNAs terminated by hairpin telomeres from replicated intermediates in bacteria that possess linear replicons. The telomere resolvase of Agrobacterium tumefaciens, TelA, has been examined at the structural and biochemical level. The N-terminal domain of TelA, while not required for telomere resolution, has been demonstrated to play an autoinhibitory role in telomere resolution, conferring divalent metal responsiveness on the reaction. The N-terminal domain also inhibits the competing reactions of hp telomere fusion and recombination between replicated telomere junctions. Due to the absence of the N-terminal domain from TelA/DNA co-crystal structures we produced an AlphaFold model of a TelA monomer. The AlphaFold model suggested the presence of two inhibitory interfaces; one between the N-terminal domain and the catalytic domain and a second interface between the C-terminal helix and the N-core domain of the protein. We produced mutant TelA's designed to weaken these putative interfaces to test the validity of the modeled interfaces. While our analysis did not bear out the details of the predicted interfaces the model was, nonetheless, extremely useful in guiding design of mutations that, when combined, demonstrated an additive activation of TelA exceeding 250-fold. For some of these hyperactive mutants stimulation of telomere resolution has also been accompanied by activation of competing reactions. However, we have also characterized hyperactive TelA mutants that retain enough autoinhibition to suppress the competing reactions.
端粒酶是一类 DNA 切割和连接酶家族，它们从具有线性复制子的细菌中的复制中间体产生线性 DNA，这些线性 DNA 由发夹状端粒末端。农杆菌的端粒酶 TelA 在结构和生化水平上进行了研究。虽然 TelA 的 N 端结构域不是端粒分解所必需的，但已证明它在端粒分解中发挥自动抑制作用，使反应对二价金属有响应。N 端结构域还抑制 hp 端粒融合和复制端粒连接之间的竞争反应。由于我们生成的 TelA/DNA 共晶结构中没有 N 端结构域，因此我们生成了 TelA 单体的 AlphaFold 模型。AlphaFold 模型表明存在两个抑制界面；一个在 N 端结构域和催化结构域之间，另一个在 C 端螺旋和蛋白质的 N 核心结构域之间。我们生成了设计用于削弱这些假定界面的突变体 TelA，以测试模型界面的有效性。虽然我们的分析没有证实预测界面的细节，但该模型非常有用，可以指导设计突变，当这些突变组合在一起时，TelA 的激活超过 250 倍。对于其中一些超活性突变体，端粒分解的刺激也伴随着竞争反应的激活。然而，我们还表征了超活性 TelA 突变体，它们保留了足够的自动抑制作用以抑制竞争反应。
