DNA polymerase δ stalls on telomeric lagging strand templates independently from G-quadruplex formation

Abstract

Previous evidence indicates that telomeres resemble common fragile sites and present a challenge for DNA replication. The precise impediments to replication fork progression at telomeric TTAGGG repeats are unknown, but are proposed to include G-quadruplexes (G4) on the G-rich strand. Here we examined DNA synthesis and progression by the replicative DNA polymerase δ/proliferating cell nuclear antigen/replication factor C complex on telomeric templates that mimic the leading C-rich and lagging G-rich strands. Increased polymerase stalling occurred on the G-rich template, compared with the C-rich and nontelomeric templates. Suppression of G4 formation by substituting Li(+) for K(+) as the cation, or by using templates with 7-deaza-G residues, did not alleviate Pol δ pause sites within the G residues. Furthermore, we provide evidence that G4 folding is less stable on single-stranded circular TTAGGG templates where ends are constrained, compared with linear oligonucleotides. Artificially stabilizing G4 structures on the circular templates with the G4 ligand BRACO-19 inhibited Pol δ progression into the G-rich repeats. Similar results were obtained for yeast and human Pol δ complexes. Our data indicate that G4 formation is not required for polymerase stalling on telomeric lagging strands and suggest that an alternative mechanism, in addition to stable G4s, contributes to replication stalling at telomeres.

Figure 1.

Templates for DNA synthesis. (A) The control, telomeric leading strand or telomeric lagging strand templates were generated by annealing a primer to circular ssDNA. Primers created either a 3- (black line) or 6- (gray line) nt RS before telomeric repeat residues (underlined nucleotides). Sequences of the first 66 template nucleotides are shown 5′ to 3′. (B) Schematic of potential secondary structures in TTAGGG repeats. Hairpin and triplex structures are proposed intermediates in G4 folding. Monovalent ions K⁺ or Na⁺ (gray balls) stabilize the G4.

Figure 2.

Pol δ stalls at TTAGGG repeats on lagging strand templates. Reactions contained 6.6 nM circular ssDNA primed with a ³²P end-labeled 40-mer to allow a 3-nt RS before telomeric sequence (see Figure 1). Reactions were initiated by adding 6.6 nM Pol δ and were incubated under standard conditions and 90 mM NaCl at 30°C. Aliquots were terminated at 5, 15 and 30 min and run on 10% polyacrylamide denaturing gels. (A) Gel images for reactions containing Pol δ alone or for templates preincubated with 20 nM PCNA and 13 nM RFC. Thick black lines mark locations of the telomeric repeats, and Gs mark the third guanine of each repeat. RS indicates the 3-nt RS, and P marks the primer. (B) Prominent stall sites occurred at the third Gs in the TTAGGG repeats on the template; marked with an arrow.

Figure 3.

Preventing G4 folding on telomeric lagging strand templates does not suppress Pol δ stalling. Reactions contained 6.6 nM of circular ssDNA templates containing 10 (A–B) or 4 (C and Supplementary Figure S2) TTAGGG repeats, primed with a 40-mer for a 3-nt RS. Primers were annealed in either 100 mM KCl or 100 mM LiCl. Reactions were initiated by adding 6.6 nM Pol δ alone or with 20 nM PCNA and 13 nM RFC, and were conducted under standard conditions except NaCl was substituted with 100 mM KCl or 100 mM LiCl. Aliquots were terminated at 5, 15 and 30 min and run on denaturing gels. Thick lines mark the telomeric repeats. RS indicates the running start, P marks the primer and Gs indicate the third guanine of each repeat. (B–C) Quantitation of Pol δ/PCNA/RFC reactions on (TTAGGG)₁₀ (B) or (TTAGGG)₄ (C) templates. The percent of primers extended past the RS were calculated as a function of total DNA. The percent of products terminated within the RS nucleotides were calculated as a function of total DNA. K⁺, black line and squares; Li⁺, gray line and circles. Mean and standard deviations from two to three independent experiments.

Figure 4.

Pol δ exhibits increased blockage on linear templates with 4 TTAGGG repeats compared with 10 in the presence of KCl. (A) Reactions contained 6.6 nM linear ssDNA with 10 or 4 (TTAGGG) repeats, four repeats with two 7-deazagaunine substitutions (indicated by asterisk), or a nontelomeric sequence (Supplementary Table S1). Templates were annealed with a primer in either 100 mM KCl or LiCl, for a 6-nt RS (see schematic). Reactions were initiated with 0.41, 1.6 or 6.6 nM Pol δ and were incubated under standard conditions except NaCl was replaced with 100 mM KCl or LiCl. Reactions were terminated at 30 min and run on denaturing gels. Thick lines mark repeats, RS marks the first 6 nt, P indicates primers and Gs mark the third guanine of each repeat. (B) The percent of products terminated within the RS was calculated as a function of total DNA. K⁺, black line and squares; Li⁺, gray line and circles. Mean and standard deviations from two to three independent experiments.

Figure 5.

A greater fraction of linear templates exhibit stable G4 structures compared with circular templates. Representative AFM images of circular gapped DNA substrates with (TTAGGG)₁₀ ssDNA (A) or CCTAA(CCCTAA)₉C ssDNA (B) regions. The image is 1 × 1 μm at 2 nm Z-scale. The white arrow points to a characteristic G4 structure (height = 1.2 nm) on a circular (TTAGGG)₁₀ gapped substrate.

Figure 6.

Pol δ stalling within G-rich telomeric repeats is conserved. Reactions contained 6.6 nM ssDNA primed with a 40-mer for a 3-nt RS (see Figure 1). Reactions were initiated by adding 10 nM human Pol δ and were incubated under standard conditions with 5 mM NaCl at 37°C. Aliquots were terminated at 5, 15 and 30 min and run on denaturing gels. Gel images are shown for reactions containing Pol δ alone or for templates preincubated with 170 nM human PCNA and 13 nM RFC as indicated. Thick lines indicate telomeric repeats, P marks the primer, RS indicates the running start and Gs or Cs indicate the third guanine or cytosine, respectively, in each repeat.

Figure 7.

G4 stabilizer BRACO-19 increases Pol δ stalling and blockage on TTAGGG templates in the presence of K⁺. (A) The (TTAGGG)₁₀ circular ssDNA substrate was annealed in either 100 mM KCl or LiCl with a primer for a 3-nt RS. Polymerase reactions contained 6.6 nM templates preincubated with 0, 0.16, 0.32, 0.80, 4.0 or 8.0 µM BRACO-19, 170 nM hPCNA and 13 nM RFC. Reactions were initiated by adding 10 nM hPol δ, and were conducted in standard conditions except NaCl was substituted with either 30 mM KCl or LiCl. Aliquots were terminated at 5, 15 and 30 min and run on denaturing gels. Thick lines indicate telomeric repeats, P marks the primer, RS indicates the running start and Gs indicate the third guanine in each repeat. Asterisk indicates shift to pause sites at TA sites. (B) The percent of primers extended past the RS. (C) The percent of products terminated within the three RS. (D) The percent of unextended primers. K⁺, black line and squares; Li⁺, gray line and circles. Data are mean and standard deviations from two to three independent experiments.