Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA.
Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH 44106, USA; Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA.
J Mol Biol. 2021 Apr 2;433(7):166846. doi: 10.1016/j.jmb.2021.166846. Epub 2021 Feb 4.
Chromosome ends are protected by guanosine-rich telomere DNA that forms stable G-quadruplex (G4) structures. The heterodimeric POT1-TPP1 complex interacts specifically with telomere DNA to shield it from illicit DNA damage repair and to resolve secondary structure that impedes telomere extension. The mechanism by which POT1-TPP1 accomplishes these tasks is poorly understood. Here, we establish the kinetic framework for POT1-TPP1 binding and unfolding of telomere G4 DNA. Our data identify two modes of POT1-TPP1 destabilization of G4 DNA that are governed by protein concentration. At low concentrations, POT1-TPP1 passively captures transiently unfolded G4s. At higher concentrations, POT1-TPP1 proteins bind to G4s to actively destabilize the DNA structures. Cancer-associated POT1-TPP1 mutations impair multiple reaction steps in this process, resulting in less efficient destabilization of G4 structures. The mechanistic insight highlights the importance of cell cycle dependent expression and localization of the POT1-TPP1 complex and distinguishes diverse functions of this complex in telomere maintenance.
染色体末端由富含鸟嘌呤的端粒 DNA 保护，这些 DNA 形成稳定的 G-四链体 (G4) 结构。异二聚体 POT1-TPP1 复合物特异性地与端粒 DNA 相互作用，保护其免受非法的 DNA 损伤修复，并解决阻碍端粒延伸的二级结构。POT1-TPP1 完成这些任务的机制尚不清楚。在这里，我们建立了 POT1-TPP1 结合和解折叠端粒 G4 DNA 的动力学框架。我们的数据确定了两种 POT1-TPP1 破坏 G4 DNA 的模式，这两种模式受蛋白质浓度的控制。在低浓度下，POT1-TPP1 被动捕获瞬时展开的 G4。在较高浓度下，POT1-TPP1 蛋白与 G4 结合以主动破坏 DNA 结构。与癌症相关的 POT1-TPP1 突变会损害该过程中的多个反应步骤，导致 G4 结构的去稳定化效率降低。这种机制上的见解强调了 POT1-TPP1 复合物在细胞周期依赖性表达和定位中的重要性，并区分了该复合物在端粒维持中的不同功能。
