Human POT1 Unfolds G-Quadruplexes by Conformational Selection

Abstract

The reaction mechanism by which shelterin protein POT1 (Protection of Telomeres) unfolds human telomeric G-quadruplex structures is not fully understood. We report here kinetic, thermodynamic, hydrodynamic and computational studies that show that a conformational selection mechanism, in which POT1 binding is coupled to an obligatory unfolding reaction, is the most plausible mechanism. We show that binding of the single-strand oligonucleotide d[TTAGGGTTAG] to POT1 is fast, with an apparent relaxation time of 80.0 ± 0.4 ms, and strong, with a binding free energy of -10.1 ± 0.3 kcal mol -1 . That favourable free energy arises from a large favourable enthalpy contribution of -38.2 ± 0.3 kcal mol -1 . In sharp contrast, the binding of POT1 to an initially folded 24 nt G-quadruplex structure is slow, with an average relaxation time of 2000-3000 s. Fluorescence, circular dichroism and analytical ultracentrifugation studies show that POT1 binding is coupled to quadruplex unfolding with a final stoichiometry of 2 POT1 molecules bound per 24 nt DNA. The binding isotherm for the POT1quadruplex binding interaction is sigmoidal, indicative of a complex reaction. A conformational selection model that includes equilibrium constants for both G-quadruplex unfolding and POT1 binding to the resultant single-strand provides an excellent quantitative fit to the experimental binding data. The overall favourable free energy of the POT1-quadruplex interaction is -7.1 kcal mol -1 , which arises from a balance between unfavourable free energy of +3.4 kcal mol -1 for quadruplex unfolding and a large, favorable free energy of -10.5 kcal mol -1 for POT1 binding. We show that POT1 can unfold and bind to any conformational form of human telomeric G-quadruplex (antiparallel, hybrid or parallel), but will not interact with duplex DNA or with a parallel G-quadruplex structure formed by a c-myc promoter sequence. Finally, molecular dynamics simulations provide a detailed structural model of a 2:1 POT1:DNA complex that is fully consistent with experimental biophysical results.

A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution

“…Previous studies with relatively unstable antiparallel telomeric G4s in Na + have observed passive trapping of the unfolded G4 by a peptide nucleic acid (PNA) oligonucleotide; the unfolding was zero order with respect to the PNA molecule, suggesting that the initial step is a rate-determining internal rearrangement of the quadruplex (Green et al, 2003). In contrast, both parallel G4s used in our experiments were highly stable; neither demonstrated any spontaneous unwinding during the observation time of the smFRET experiments, and in our previous work we have shown that the parallel tetrameric G4 did not show any unwinding over the time scale of hours (Moye et al, 2015).Telomeric protein POT1 unwinds a G4 through a conformational selection mechanism in which G4 unwinding occurs prior to POT1 binding (Chaires et al, 2020); each of the OB folds of two POT1 molecules then binds to one telomeric repeat of the 4-repeat G4 in a stepwise manner (Hwang et al, 2012). For telomerase, we favour a model in which the enzyme binds the G4 while it is still structured, followed by invasion by the RNA template; confirmation of this model will require further kinetic analyses.…”