title: 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. Protection of telomeres 1 (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 fivefold higher than for G-quadruplex structures formed in the presence of K(+). However, the binding of the 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 the 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.
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Mullins
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Malligarjunan
authors.initials: M
authors.lastname: Rajavel
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Wilnelly
authors.initials: W
authors.lastname: Hernandez-Sanchez
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: de la Fuente
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Sherri M
authors.initials: SM
authors.lastname: Biendarra
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Harris
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA; Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA. Electronic address: derek.taylor@case.edu.
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Taylor
mesh_terms: D000626:Aminopeptidases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000378:metabolism*; D054856:G-Quadruplexes; D006801:Humans; D011110:Polymorphism, Genetic / Q000235:genetics*; D011485:Protein Binding; D057057:Serine Proteases / Q000378:metabolism*; D000089804:Shelterin Complex / Q000378:metabolism*; D019098:Telomerase / Q000378:metabolism; D016615:Telomere / Q000378:metabolism*; D034501:Telomere-Binding Proteins / Q000378:metabolism*
chemical_list: C495876:ACD protein, human; D004277:DNA, Single-Stranded; C440849:POT1 protein, human; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins; D019098:Telomerase; D057057:Serine Proteases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases
keywords: G-quadruplex; shelterin; telomerase; telomere