Polymerase γ efficiently replicates through many natural template barriers but stalls at the HSP1 quadruplex

Abstract

Faithful replication of the mitochondrial genome is carried out by a set of key nuclear-encoded proteins. DNA polymerase γ is a core component of the mtDNA replisome and the only replicative DNA polymerase localized to mitochondria. The asynchronous mechanism of mtDNA replication predicts that the replication machinery encounters dsDNA and unique physical barriers such as structured genes, G-quadruplexes, and other obstacles. In vitro experiments here provide evidence that the polymerase γ heterotrimer is well-adapted to efficiently synthesize DNA, despite the presence of many naturally occurring roadblocks. However, we identified a specific G-quadruplex-forming sequence at the heavy-strand promoter (HSP1) that has the potential to cause significant stalling of mtDNA replication. Furthermore, this structured region of DNA corresponds to the break site for a large (3,895 bp) deletion observed in mitochondrial disease patients. The presence of this deletion in humans correlates with UV exposure, and we have found that efficiency of polymerase γ DNA synthesis is reduced after this quadruplex is exposed to UV in vitro.

Keywords: DNA polymerase γ; DNA replication; DNA structure; G-quadruplex; heavy-strand promoter; mitochondrial DNA (mtDNA); mitochondrial DNA damage; mtDNA deletion.

Figure 1.

Pol γ primer extension with structured oligo templates. A, illustrative denaturing PAGE gel slices, at time = 0 or 10 min, showing Pol γ catalytic subunit pausing to form intermediate (left) or Pol γ heterotrimer catalyzing only full-length primer extension (right) during primer extension across the OriL template oligo. Full-time course gel is shown in Fig. S1. B, primer extension time course for Pol γ catalytic subunit alone. Substrate template strand is an OriL mimic capable of adopting a stem-loop fold. Formation of reaction intermediates (red) and full-length product (orange) are shown. Reactions were carried out in 100 mm NaCl (closed symbols) and 200 mm NaCl (open symbols). In low salt, p140 (30 nm) catalyzed primer extension to form intermediate species at an initial rate of 0.51 nm/s and full-length product at 0.12 nm/s. At high salt, p140 catalyzed extension to intermediate-length products at 0.04 nm/s. C, primer extension time courses as described in (B) for the Pol γ heterotrimer with an OriL substrate template. The Pol γ complex catalyzed primer extension to produce full-length products in low and high salt at 0.09 nm/s and 0.12 nm/s, respectively. D, schematic representation of tRNA-coding oligo substrates, with Pol γ shown as a green box. The mtDNA gene encoding tRNA-Pro is templated on the light strand and coded by the heavy strand. E and F, reaction progress curves for Pol γ-catalyzed primer extension using either the catalytic subunit alone (E) or the heterotrimer (F) and the tRNA-Pro oligos. Synthesis of the mtDNA light strand is denoted by open symbols, whereas heavy-strand synthesis is denoted by closed symbols. Red circles represent formation of intermediate species and orange squares represent formation of full-length product. When synthesizing L-strand (E), p140 alone (5 nm) catalyzed primer extension to form intermediate species at a rate of 0.02 nm/s and full-length product at 0.08 nm/s. Alternatively, during H-strand synthesis, p140 alone catalyzed primer extension to full-length product at a rate of 0.17 nm/s. The Pol γ complex (F) extended primers synthesizing both L-strand and H-strand to full-length products at 0.02 nm/s and 0.04 nm/s, respectively. Error bars, when larger than data symbols, represent standard error of two replicates.

Figure 2.

Displacement of downstream oligonucleotides. A, hairpin substrate used to measure the strand displacement activity of Pol γ. The red oligo is a Cy5-labeled 100-mer hairpin and provides both the template and the 3′-OH site for Pol γ extension. The green strand is a 25-mer RNA with an internal Cy3 label. The blue strand, which anneals immediately adjacent to the RNA, is a 28-mer DNA with an internal FAM label. The three fluorophores excite at unique wavelengths, allowing for visualization of each oligo component after PAGE. B, reaction progress curves showing activity of Pol γ catalytic subunit alone (closed circles) and heterotrimer (open circles) on the hairpin substrate lacking downstream oligonucleotides. Pol γ catalyzes primer extension to full-length product. C and D, reaction progress curves for Pol γ heterotrimer with the hairpin substrate and either the downstream 28-mer DNA (C) or 25-mer RNA (D). Displacement of the downstream oligo is represented by the matching colored symbols (displaced DNA is blue and displaced RNA is green. Red symbols represent products from gap-filling that stalled at the downstream oligo). E, reaction progress curves for Pol γ activity on the hairpin substrate with both downstream oligos annealed. Closed circles represent activity by the Pol γ catalytic subunit alone and open diamonds represent the heterotrimer. Red symbols correspond to intermediate products formed through gap-filling activity, green symbols correspond to higher molecular weight intermediate products formed through displacement of the RNA strand, and blue symbols correspond to full-length products formed through displacement of both RNA and DNA strands. In all graphs, error bars, when larger than data symbols, represent standard error of up to six replicates in (B), triplicates in (C) and (D), and duplicates in (E).

Figure 3.

Primer extension by Pol γ pauses at G4 structures. A, time course of primer extension by Pol γ heterotrimer on the G4_Neg substrate was assessed by denaturing PAGE. B and C, activity of the Pol γ catalytic subunit alone and the Pol γ heterotrimer, respectively, on the G4_Neg oligo substrate. Pol γ catalyzes primer extension to form only full-length product (orange squares). No intermediate species are observed (red circles). D, time course of primer extension by Pol γ heterotrimer with the G4_Cntrl substrate was assessed by denaturing PAGE. Both intermediate-length and full-length products are observed. E and F, activity of the Pol γ catalytic subunit alone and the Pol γ heterotrimer, respectively, on the G4_Cntrl oligo substrate. Pol γ catalyzes primer extension to form both full-length product (orange squares) and intermediate species (red circles). Total primer extension of both intermediate- and full-length products is shown (green diamonds).

Figure 4.

G4 substrate library. MtDNA was screened for potential G-quadruplex structures with the G4 Hunter and QGRS mapping algorithms. Eight specific G-quadruplex–forming sequences were selected as substrates to assess primer extension activity of Pol γ in vitro. A, mapped location of the eight mtDNA sequences chosen as quadruplex formations. B, schematic of the library design for G4 substrates. The substrates share a common primer/template combination. The primer (red) has a 5′ Cy5 label. The conserved template (black) is extended by a 9-nt spacer (orange), separate mtDNA sequences containing putative G-quadruplexes (green), and a 6-nt flanking tail (orange).

Figure 5.

Pol γ catalytic activity with G4_5 substrate is salt-dependent. A, sample PAGE gel depicting a Pol γ time course from left to right. The Pol γ heterotrimer was used for this time course, with salt at 200 mm. Substrate is primarily extended to the intermediate species, though it occasionally reaches full-length product. B, initial rates for formation of the intermediate (Vo Int., closed symbols) and full-length product (Vo Prod., open symbols) by either the isolated catalytic subunit (red circles) or the heterotrimeric Pol γ complex (orange squares) are plotted.

Figure 6.

Schematic of G4_5 positioned at UV breakpoint. A, portion of mtDNA that has been reported to be deleted in UV-exposed human skin samples. Mapped along the deletion are the genes lost. The deletion is flanked by 13-bp repeats (orange). The quadruplex formed by G4_5 is shown in purple. B, predicted mixed topology structure of G4_5 based on CD spectra. The 13-bp repeat, shown in orange, overlaps with the first and second stretch of tetrad guanines. In our in vitro experiments, DNA synthesis by Pol γ (green) pauses at the G-quadruplex structure. HSP1 corresponds to nucleotides 561–570, where position 561 is the 5′-end of the transcript.

Figure 7.

UV irradiation of G4_5 exacerbates stalling by Pol γ. The G4_5 quadruplex substrate was exposed to UV radiation, as described under “Experimental procedures.” A, the activity of Pol γ complex (p140+p55) on the G4_5 quadruplex substrates that had been either exposed to 10,000 J/m² (+UV) or shielded from (Dark) 254 nm UV light was assessed in primer extension reactions. Products generated as a function of time were analyzed by denaturing PAGE. The positions of substrate, full-length product, and three main regions of intermediate-length products (intermediates A, B, and C) are labeled. B, the sum of all intermediate- and full-length product species generated at the indicated reaction times from +UV (red) and dark (orange) G4_5 quadruplex substrates was quantified. C, intermediate species formed by Pol γ on G4_5 quadruplex substrates that were exposed to (red) or shielded from (orange) UV were quantified for intermediates A (circles), B (squares), and C (diamonds) for the indicated reaction times. D, full-length product species formed by Pol γ on G4_5 quadruplex substrates that were shielded (orange squares) or exposed to either 10,000 J/m² (red circles) or 30,000 J/m² (blue triangles) of UV irradiation were quantified for the indicated reaction times. Error bars, when larger than data symbols, represent standard error of two replicates.