POT1–TPP1 Binding and Unfolding of Telomere DNA Discriminates against Structural Polymorphism

Abstract

Telomeres are nucleoprotein complexes that reside at the ends of linear chromosomes and help maintain genomic integrity. POT1 and TPP1 are telomere-specific proteins that bind as a heterodimer to single-stranded telomere DNA to prevent illicit DNA damage responses and to enhance telomerase-mediated telomere extension. Telomere DNA is guanosine-rich and, as such, can form highly stable secondary structures including G-quadruplexes. G-quadruplex DNA folds into different topologies that are determined by several factors including monovalent ion composition and the precise sequence and length of the DNA. Here, we explore the influence of DNA secondary structure on POT1-TPP1 binding. Equilibrium binding assays reveal that the POT1-TPP1 complex binds G-quadruplex structures formed in buffers containing Na+ with an affinity that is 5-fold higher than for G-quadruplex structures formed in the presence of K+. However, binding of a second heterodimer is insensitive to DNA secondary structure, presumably due to unfolding resulting from binding of the first POT1-TPP1. We further show that the rate constant for POT1-TPP1-induced unfolding of DNA secondary structure is substantially faster for G-quadruplex topologies formed in the presence of Na+ ions. When bound to DNA, POT1-TPP1 forms complexes with similar CD spectra and enhances telomerase activity for all DNA substrates tested, regardless of substrate secondary structure or solution monovalent ion composition. Together, these data indicate that binding of POT1-TPP1 unfolds telomere secondary structure to assist loading of additional heterodimers and to ensure efficient promotion of telomerase-mediated extension.

Administration of a Nucleoside Analog Promotes Cancer Cell Death in a Telomerase-Dependent Manner

“…Human full-length POT1 and TPP1 were co-expressed in Spodoptera frugiperda 9 (Sf9) insect cells, using a recombinant baculovirus expression system as previously described (Mullins et al, 2016). Briefly, Sf9 cells expressing full-length POT1 and N-terminal 6 His-tagged TPP1 (89-334) were lysed in a lysis buffer containing 25 mM HEPES (pH 8.0), 300 mM NaCl, 5 mM DTT, 5 mM benzamidine, 1 mM PMSF, and 1 complete ULTRA protease inhibitor cocktail tablet (Roche Diagnostics, Indianapolis, IN, USA).…”

“…A telomerase extension assay was performed to identify non-native nucleotide analogs that can be incorporated into the telomeric DNA, as described previously (Mullins et al, 2016). Briefly, human (h)TERT and hTR were co-transfected into HEK293T cells.…”

“…Quantification of in vitro telomerase assay products was performed as described previously (Mullins et al, 2016). Briefly, relative intensities for each hexamer repeat were calculated and normalized against the loading control for each lane.…”

Human POT1 Unfolds G-Quadruplexes by Conformational Selection

“…Finally, a study from the Taylor laboratory (20) used an electrophoretic mobility shift assay to monitor the interaction of a POT1-TPP1 complex with a variety of telomeric G4 structures. A sequential two-step binding model was used to analyse their binding isotherms, and circular dichroism was used to show that the initially folded G4 structure was disrupted upon binding.…”

Dynamic peptides of human TPP1 fulfill diverse functions in telomere maintenance

“…Furthermore, TPP1 actively recruits telomerase to the telomere and functions as a telomerase processivity factor when coupled with the POT1 protein (18,23,28,29). Finally, when combined with POT1, TPP1 aids in disrupting secondary structures, including G-quadruplexes that are intrinsic to G-rich telomere DNA (30–32) and restricts telomere resection after DNA replication (33,34). Despite an appreciable amount of data focused on TPP1 function, however, it remains unclear as to how this protein can regulate so many different processes and what molecular alterations govern the switch from one action to another.…”