Human POT1 unfolds G-quadruplexes by conformational selection

Abstract

The reaction mechanism by which the shelterin protein POT1 (Protection of Telomeres 1) 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. Stopped-flow kinetic and spectroscopic titration studies, along with isothermal calorimetry, were used to show that binding of the single-strand oligonucleotide d[TTAGGGTTAG] to POT1 is both fast (80 ms) and strong (−10.1 ± 0.3 kcal mol−1). In sharp contrast, kinetic studies showed the binding of POT1 to an initially folded 24 nt G-quadruplex structure is four orders of magnitude slower. Fluorescence, circular dichroism and analytical ultracentrifugation studies showed that POT1 binding is coupled to quadruplex unfolding, with a final complex with a stoichiometry of 2 POT1 per 24 nt DNA. The binding isotherm for the POT1-quadruplex interaction was 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 provided an excellent quantitative fit to the experimental binding data. POT1 unfolded and bound to any conformational form of human telomeric G-quadruplex (antiparallel, hybrid, parallel monomers or a 48 nt sequence with two contiguous quadruplexes), but did not avidly interact with duplex DNA or with other G-quadruplex structures. Finally, molecular dynamics simulations provided a detailed structural model of a 2:1 POT1:DNA complex that is fully consistent with experimental biophysical results.

G-Quadruplexes at Telomeres: Friend or Foe?

“…The potential formation of G-quadruplexes at overhangs would therefore be in competition with POT1 binding; indeed, POT1 has been shown to unwind intramolecular G-quadruplexes in order to bind to telomeric DNA [ 143 , 144 ]. POT1 unwinds G-quadruplexes through a conformational selection mechanism in which G-quadruplex unwinding occurs prior to POT1 binding [ 145 ]; each of the OB folds of two POT1 molecules then binds to one telomeric repeat of the 4-repeat G-quadruplex in a stepwise manner ( Figure 5 ) [ 146 , 147 ]. Another G4-unwinding protein that is known to localize to telomeres is replication protein A (RPA) [ 148 ], which plays a major role in activating the DNA damage response to single-stranded DNA [ 149 ].…”

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.…”

Antisense Oligonucleotides Used to Identify Telomeric G-Quadruplexes in Metaphase Chromosomes and Fixed Cells by Fluorescence Lifetime Imaging Microscopy of o-BMVC Foci

“…Notably, the importance of telomeres is not only the formation of G4 structures, but also the interaction with the cancer-associated enzyme telomerase [ 14 , 56 , 57 ]. Given that the protection of telomeres 1 (POT1) protein can unfold G4 structure prior to POT1 binding [ 42 , 51 , 58 ], visualizing how antisense sequences affect the protein binding to telomeric DNA could provide valuable insights into G4-protein interaction, for increasing our understanding of telomere biology.…”