title: Coordinated interactions of multiple POT1-TPP1 proteins with telomere DNA.
abstract: Telomeres are macromolecular nucleoprotein complexes that protect the ends of eukaryotic chromosomes from degradation, end-to-end fusion events, and from engaging the DNA damage response. However, the assembly of this essential DNA-protein complex is poorly understood. Telomere DNA consists of the repeated double-stranded sequence 5'-TTAGGG-3' in vertebrates, followed by a single-stranded DNA overhang with the same sequence. Both double- and single-stranded regions are coated with high specificity by telomere end-binding proteins, including POT1 and TPP1, that bind as a heterodimer to single-stranded telomeric DNA. Multiple POT1-TPP1 proteins must fully coat the single-stranded telomere DNA to form a functional telomere. To better understand the mechanism of multiple binding, we mutated or deleted the two guanosine nucleotides residing between adjacent POT1-TPP1 recognition sites in single-stranded telomere DNA that are not required for multiple POT1-TPP1 binding events. Circular dichroism demonstrated that spectra from the native telomere sequence are characteristic of a G-quadruplex secondary structure, whereas the altered telomere sequences were devoid of these signatures. The altered telomere strands, however, facilitated more cooperative loading of multiple POT1-TPP1 proteins compared with the wild-type telomere sequence. Finally, we show that a 48-nucleotide DNA with a telomere sequence is more susceptible to nuclease digestion when coated with POT1-TPP1 proteins than when it is left uncoated. Together, these data suggest that POT1-TPP1 binds telomeric DNA in a coordinated manner to facilitate assembly of the nucleoprotein complexes into a state that is more accessible to enzymatic activity.
authors.affiliation: Department of Pharmacology, Cleveland, Ohio 44106.
authors.forename: Mark
authors.initials: M
authors.lastname: Corriveau
authors.affiliation: Department of Pharmacology, Cleveland, Ohio 44106.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Mullins
authors.affiliation: Department of Pharmacology, Cleveland, Ohio 44106.
authors.forename: Diane
authors.initials: D
authors.lastname: Baus
authors.affiliation: Department of Biochemistry, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Harris
authors.affiliation: Department of Pharmacology, Cleveland, Ohio 44106; Department of Biochemistry, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106. Electronic address: derek.taylor@case.edu.
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Taylor
mesh_terms: D002877:Chromosomes, Human / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D000089804:Shelterin Complex; D016615:Telomere / Q000737:chemistry* / Q000378:metabolism; D034501:Telomere-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C495876:ACD protein, human; D004277:DNA, Single-Stranded; C440849:POT1 protein, human; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins
keywords: DNA Binding Protein; DNA Structure; G-quadruplex; Nucleotide; POT1; TPP1; Telomerase; Telomeres