Human POT1 disrupts telomeric G-quadruplexes allowing telomerase extension in vitro - PubMed (original) (raw)

Human POT1 disrupts telomeric G-quadruplexes allowing telomerase extension in vitro

Arthur J Zaug et al. Proc Natl Acad Sci U S A. 2005.

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.

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Figures

Fig. 1.

Fig. 1.

Poor primers for telomerase are rescued by hPOT1. (A) Primer a extended by telomerase, which incorporates nucleotides shown as lowercase letters; hPOT1 binds to either of two overlapping binding sites on primer a (Left). Sequence of primers used in this study (Right). Nontelomeric nucleotides are underlined, blocks of Gs are highlighted with bars. (B) Direct telomerase-activity assay performed with 100 nM primer G-a (lanes 1-3), GG-a (lanes 4-6), or GGG-a (lanes 7-9), in the presence of protein buffer (-) or 333 nM hPOT1 (+). Lanes 0, the telomerase assay was performed with immunopurified hTERT without the RNA (hTER) subunit. (C) Extension of primers in the presence and absence of hPOT1 and in the presence and absence of 50 mM KCl. Concentrations of primer and protein are as in A. M and M′, markers synthesized by using the direct telomerase activity assay with primer d(GGTTAG)3 in the presence of dGTP only (+2) or in the presence of dGTP and dTTP (+4). LC, loading control.

Fig. 2.

Fig. 2.

Poor primers form intramolecular G-quadruplexes. (A) Native gel analysis of primers. (B) Direct telomerase activity assay using the same primers (100 nM). Lanes M and M′ are as described in Fig. 1.

Fig. 3.

Fig. 3.

hPOT1 forms stable complexes with G-quadruplex-forming primers. (A) Native gel analysis of 100 nM primers in the presence (+) or absence (-)of333 nM hPOT1. (B) Denaturing gel electrophoresis of SVPI digests of primers alone (lanes 1-4), in complex with hPOT1 (lanes 5-8), or in complex with antisense DNA (lanes 9-12); no SVPI, lanes 13-16. (C) Native gel analysis of the primers (100 nM) in the presence (+) or absence (-) of 500 nM antisense DNA. The antisense DNA sequence is written 3′-to-5′. (D) Direct telomerase activity assays. Before adding telomerase, the DNA oligonucleotides (100 nM) were incubated with 500 nM antisense DNA for 10 min at room temperature. Lanes M and M′ are as described in Fig. 1.

Fig. 4.

Fig. 4.

Model for hPOT1 disruption of intramolecular G-quadruplex DNAs, allowing their extension by telomerase. Quadruplex form is shown in equilibrium with partially or completely unstructured forms, which bind hPOT1. Our data do not rule out a more active opening of the quadruplex structure by hPOT1, indicated by?. When hPOT1 binds near the 5′ end of the primer, leaving an 8-nt tail, it can be extended by telomerase (10). When hPOT1 binds near the 3′ end of the primer, leaving a 2-nt tail, there is no reaction (N.R.).

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