Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dow Street, Dundee DD1 5EH, UK.
Nucleic Acids Res. 2018 Nov 16;46(20):11089-11098. doi: 10.1093/nar/gky863.
GEN1 is a member of the FEN/EXO family of structure-selective nucleases that cleave 1 nt 3' to a variety of branchpoints. For each, the H2TH motif binds a monovalent ion and plays an important role in binding one helical arm of the substrates. We investigate here the importance of this metal ion on substrate specificity and GEN1 structure. In the presence of K+ ions the substrate specificity is wider than in Na+, yet four-way junctions remain the preferred substrate. In a combination of K+ and Mg2+ second strand cleavage is accelerated 17-fold, ensuring bilateral cleavage of the junction. We have solved crystal structures of Chaetomium thermophilum GEN1 with Cs+, K+ and Na+ bound. With bound Cs+ the loop of the H2TH motif extends toward the active site so that D199 coordinates a Mg2+, buttressed by an interaction of the adjacent Y200. With the lighter ions bound the H2TH loop changes conformation and retracts away from the active site. We hypothesize this conformational change might play a role in second strand cleavage acceleration.
GEN1 是 FEN/EXO 家族结构选择性核酸内切酶的成员，可切割各种分支点的 1nt3'。对于每种酶，H2TH 基序结合单价离子，并在结合底物的一条螺旋臂方面发挥重要作用。我们在这里研究该金属离子对底物特异性和 GEN1 结构的重要性。在 K+离子存在下，底物特异性比在 Na+中更宽，但四链结仍然是首选底物。在 K+和 Mg2+的组合中，第二条链的切割速度加快了 17 倍，从而确保了连接点的双侧切割。我们已经解决了与 Cs+，K+和 Na+结合的嗜热毛壳菌 GEN1 的晶体结构。与结合的 Cs+结合时，H2TH 基序的环向活性位点延伸，使得 D199 配位一个 Mg2+，由相邻的 Y200 支撑。与较轻的离子结合时，H2TH 环改变构象并从活性位点缩回。我们假设这种构象变化可能在第二条链切割加速中起作用。
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