Human POT1 disrupts telomeric G-quadruplexes allowing telomerase extension in vitro.
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.
Zaug
Arthur J
AJ
Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA.
Podell
Elaine R
ER
Cech
Thomas R
TR
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*
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