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. 2011 Nov;39(21):9238-49.
doi: 10.1093/nar/gkr653. Epub 2011 Aug 12.

The Mitochondrial DNA Helicase TWINKLE Can Assemble on a Closed Circular Template and Support Initiation of DNA Synthesis

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Free PMC article

The Mitochondrial DNA Helicase TWINKLE Can Assemble on a Closed Circular Template and Support Initiation of DNA Synthesis

Elisabeth Jemt et al. Nucleic Acids Res. .
Free PMC article

Abstract

Mitochondrial DNA replication is performed by a simple machinery, containing the TWINKLE DNA helicase, a single-stranded DNA-binding protein, and the mitochondrial DNA polymerase γ. In addition, mitochondrial RNA polymerase is required for primer formation at the origins of DNA replication. TWINKLE adopts a hexameric ring-shaped structure that must load on the closed circular mtDNA genome. In other systems, a specialized helicase loader often facilitates helicase loading. We here demonstrate that TWINKLE can function without a specialized loader. We also show that the mitochondrial replication machinery can assemble on a closed circular DNA template and efficiently elongate a DNA primer in a manner that closely resembles initiation of mtDNA synthesis in vivo.

Figures

Figure 1.
Figure 1.
TWINKLE can efficiently load onto circular DNA substrates. TWINKLE interactions with circular ssDNA, linear ssDNA and linear dsDNA. (A) TWINKLE–DNA interactions were monitored by EMSA using 32P-labeled circular ssDNA (100 nt, upper panel), linear dsDNA (100 bp, middle panel) or linear ssDNA (100 nt, lower panel) as described in ‘Materials and Methods’ section. Incubation temperatures and the amount of TWINKLE (0.1 or 0.2 pmol) are indicated at the top of the figure. Lanes 7–12 and 14–19, were cross-linked with glutaraldehyde and lanes 14–19; were further treated with SDS and heated at +95°C for 5 min. (B) The binding of TWINKLE to circular ssDNA is nucleotide independent. Binding reactions between TWINKLE and closed circular ssDNA in the absence (lanes 1–4) or presence of UTP (lanes 5–8) were performed as described in ‘Materials and Methods’ section. Lanes 1 and 5, DNA substrate alone; lanes 2–4 and 6–8, were incubated with 200 fmol TWINKLE at +4°C; lanes 3–4 and 7–8, were cross-linked with glutaraldehyde; and lanes 4 and 8 were further treated with SDS and heated at +95°C for 5 min.
Figure 2.
Figure 2.
TWINKLE can load onto a circular ssDNA and unwind duplex DNA. (A) Helicase activity of TWINKLE and T7gp4. The DNA substrates used in this study are composed of a 20-bp duplex region and a 40-nt single-stranded overhang (5′ or 3′). The helicase activity is measured as the ability of the protein to separate the annealed 60-nt radiolabeled oligonucleotide from the circular M13ssDNA. The reactions were performed at 32°C as described in ‘Materials and Methods’ section using the 5′-tailed substrate (lanes 1–9) or the 3′-tailed substrate (lanes 10–18) in the presence of 3 mM ATP and increasing amounts of TWINKLE (150, 300 and 600 fmol) or 3 mM dTTP and increasing amounts of T7gp4 (250, 500 and 1000 fmol). Lanes 1 and 10, 5′-tailed and 3′-tailed substrates heated to 100°C before loading; lanes 2 and 6, untreated 5′-tailed DNA substrate; lanes 11 and 15, untreated 3′-tailed DNA substrate; S, double-stranded substrate; P, single-stranded product. (B) Loading of TWINKLE on ssDNA in presence of UTP. Helicase activity of TWINKLE (300 fmol) was measured at 32°C using the 5′-tailed substrate (lanes 1–6) or the 3′-tailed substrate (lanes 7–12) in presence of 3 mM ATP or 3 mM UTP. Lanes 1, 4, 7 and 10, DNA substrates heated to 100°C before loading.
Figure 3.
Figure 3.
TWINKLE unwinding of a closed circular template is stimulated at higher temperatures. (A) The helicase activity of TWINKLE (300 fmol) was monitored using the 5′-tailed substrate (lanes 1–8) or the 3′-tailed substrate (lanes 9–16) in the presence of 3 mM UTP and at increasing temperatures (20°C, 25°C, 32°C, 37°C, 41°C and 45°C). After 45 min of incubation at the indicated temperature, the reaction products were separated on a 12.5% non-denaturating polyacrylamide gel. Lanes 1 and 9, DNA substrates heated at 100°C before loading; lanes 2 and 10, DNA substrate incubated at 45°C in the absence of TWINKLE; S, double-stranded substrate; P, single-stranded product. (B) Helicase assays were performed using the 5′-tailed or the 3′-tailed substrates in the presence of 200 fmol of TWINKLE and 3 mM of the indicated nucleotide at 25°C (upper panel) or at 42°C (lower panel). (C) Hydrolysis of different nucleotides by TWINKLE were measured as described in ‘Materials and Methods’ section. The reactions were performed in the presence of 200 fmol of TWINKLE, 0.5 mM of the indicated nucleotide, and in the absence (light gray and white blocks) or presence (dark gray and black bars) of M13ssDNA (188 fmol) at 25°C (light and dark gray bars) or at 42°C (white and black bars).
Figure 4.
Figure 4.
TWINKLE in combination with POLγ can support DNA replication on a circular dsDNA template. (A) The bubble template was prepared as described in ‘Materials and Methods’ section. The tentative localization of TWINKLE (light gray hexamer) and POLγ (dark gray and black heterotrimer) are indicated. The bubble region is symmetric and may contain one annealed primer on each strand, allowing initiation of DNA synthesis in opposite directions. (B) DNA replication assays were performed using the bubble template (10 fmol), POLγ (70 fmol), and increasing amounts of TWINKLE (0, 50, 200 and 750 fmol) for 60 min at 42°C. (C) DNA replication assays were performed as in (B) but in presence or absence of 750 fmol TWINKLE. The reactions were incubated for 0, 2, 15, 40 or 70 min at either 25°C (upper panel) or 42°C (lower panel). SM, size marker.
Figure 5.
Figure 5.
mtSSB does not prevent the loading of TWINKLE onto ssDNA. (A) Helicase assays were performed as described in ‘Materials and Methods’ section. Reaction mixtures contained 5 fmol of 5′-tailed or 3′-tailed DNA substrates (Figure 2A), 200 fmol of TWINKLE, and increasing amounts of mtSSB (2.5, 5 and 10 pmol of the mtSSB tetramer). The saturation levels of mtSSB relative the DNA substrates are indicated and based on the assumption that one mtSSB tetramer covers 60 nt (44). The DNA substrates were first incubated with mtSSB at room temperature for 10 min. TWINKLE was then added to the reactions and the helicase assay was performed at 42°C for 45 min in the presence of 3 mM UTP. (B) DNA replication assays were performed as described in ‘Materials and Methods’ section. The reaction mixture contained 10 fmol of the bubble template (Figure 4A), 70 fmol of POLγ, 750 fmol of TWINKLE, and increasing amounts of mtSSB (150, 300 and 600 fmol of mtSSB tetramer). The saturation levels of mtSSB relative the DNA substrates are indicated and the reactions were incubated at 37°C for 1 h.

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