title: Human POT1 disrupts telomeric G-quadruplexes allowing telomerase extension in vitro.
abstract: The POT1 (protection of telomeres 1) protein binds the ssDNA overhangs at the ends of chromosomes in diverse eukaryotes. POT1 is essential for chromosome end-protection, as best demonstrated in fission yeast. In human cells, hPOT1 is also involved in telomere-length regulation. We now show that telomeric oligonucleotides, such as d[GGG(TTAGGG)(3)], which form intramolecular G-quadruplexes through Hoogsteen base-pairing, serve as only marginal primers for extension by recombinant human telomerase; telomerase stalls after every nucleotide addition. Addition of hPOT1 to the reaction restores the normal processive elongation pattern seen with primers that cannot form G-quadruplexes. hPOT1 does not act catalytically but, instead, forms a stoichiometric complex with the DNA, freeing its 3' tail. An antisense oligonucleotide, which base-pairs near the 5' end of the telomeric sequence, leaving a telomerase-extendable 3' tail, duplicates the effect of hPOT1 on activation of G-quadruplex primers. Thus, hPOT1 may function simply by trapping the unfolded forms of these telomeric primers in an equilibrium population. We propose an additional role for hPOT1 in telomere maintenance: disrupting G-quadruplex structures in telomeric DNA, thereby allowing proper elongation by telomerase.
authors.affiliation: Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA.
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Zaug
authors.forename: Elaine R
authors.initials: ER
authors.lastname: Podell
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Cech
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000089804:Shelterin Complex; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism*; D034501:Telomere-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C440849:POT1 protein, human; D011994:Recombinant Proteins; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins; D004247:DNA; D019098:Telomerase