Human mitochondrial DNA helicase TWINKLE is both an unwinding and annealing helicase

Abstract

TWINKLE is a nucleus-encoded human mitochondrial (mt)DNA helicase. Point mutations in TWINKLE are associated with heritable neuromuscular diseases characterized by deletions in the mtDNA. To understand the biochemical basis of these diseases, it is important to define the roles of TWINKLE in mtDNA metabolism by studying its enzymatic activities. To this end, we purified native TWINKLE from Escherichia coli. The recombinant TWINKLE assembles into hexamers and higher oligomers, and addition of MgUTP stabilizes hexamers over higher oligomers. Probing into the DNA unwinding activity, we discovered that the efficiency of unwinding is greatly enhanced in the presence of a heterologous single strand-binding protein or a single-stranded (ss) DNA that is complementary to the unwound strand. We show that TWINKLE, although a helicase, has an antagonistic activity of annealing two complementary ssDNAs that interferes with unwinding in the absence of gp2.5 or ssDNA trap. Furthermore, only ssDNA and not double-stranded (ds)DNA competitively inhibits the annealing activity, although both DNAs bind with high affinities. This implies that dsDNA binds to a site that is distinct from the ssDNA-binding site that promotes annealing. Fluorescence anisotropy competition binding experiments suggest that TWINKLE has more than one ssDNA-binding sites, and we speculate that a surface-exposed ssDNA-specific site is involved in catalyzing DNA annealing. We propose that the strand annealing activity of TWINKLE may play a role in recombination-mediated replication initiation found in the mitochondria of mammalian brain and heart or in replication fork regression during repair of damaged DNA replication forks.

FIGURE 1.

A typical profile for the purification of C-His₆-TWINKLE. A, representative 4–20% SDS-PAGE shows the purification of C-terminally His-tagged TWINKLE by Ni²⁺-Sepharose chromatography and heparin-Sepharose chromatography followed by dialysis in storage buffer C (see under “Experimental Procedures”). The purification protocol has been reproduced successfully 11 times. Lane 1, molecular weight ladder (Invitrogen). Lane 2, Pre-IPTG-induced N-His₆-TWINKLE lysate. Lane 3, post-IPTG-induced N-His₆-TWINKLE lysate. Lane 4, pre-IPTG-induced C-His₆-TWINKLE lysate. Lane 5, post- IPTG-induced C-His₆-TWINKLE lysate. Lane 6, molecular weight ladder (Invitrogen). Lane 7, Ni²⁺-Sepharose affinity column eluted fraction. Lane 8, heparin-Sepharose column eluted fraction. Lane 9, heparin-Sepharose column pure fractions combined. Lane 10, sample from lane 9 dialyzed against storage buffer C. The box in lanes 2–5 highlights the induction of TWINKLE by IPTG. The arrow indicates the position of the recombinant protein for lanes 7–10 (75 kDa). B, 20% polyacrylamide/TBE gel confirms the absence of DNase contamination in the purified protein tested from 15 to 60 min at 30 °C on a 72-base DNA. Lane 1, exonuclease III + EDTA. Lane 2, exonuclease III + magnesium acetate. Lane 3, no TWINKLE + EDTA. Lane 4, no TWINKLE + magnesium acetate. Lane 5, reaction time of 15 min. Lane 6, reaction time of 30 min. Lane 7, reaction time of 60 min. Lanes 1 and 2 are the positive controls; lanes 3 and 4 are the negative controls; lanes 5–7 are reactions with TWINKLE, DNA, and magnesium acetate. The arrow indicates the smear for the digested DNA in the presence of ExoIII and Mg²⁺ in lane 2 (positive control).

FIGURE 2.

Oligomeric state of TWINKLE and effect of cofactors and DNA. A, Coomassie-stained 4–12% BisTris SDS-polyacrylamide gel showing TWINKLE cross-linking with DMS at 4 °C for 1 min (lanes 1 and 7), 8 min (lanes 2 and 8), 15 min (lanes 3 and 9), 30 min (lanes 4 and 10), and 60 min (lanes 5 and 11) in the presence (lanes 1–5) or absence (lanes 7–11) of MgUTP and in the presence of DNA. Lanes 6 and 12 are noncross-linked controls showing both monomers and very small amount of dimers. Lanes marked L are the molecular weight standards. B, Coomassie-stained 4–12% BisTris SDS-PAGE showing the DMS cross-linking of TWINKLE at 4 °C for 60 min in the presence of MgUTP in the presence and absence of DNA.

FIGURE 3.

Fluorescence anisotropy-based binding of ssDNA and dsDNA to TWINKLE. Changes in anisotropy of TMR-labeled ssDNA (A) and dsDNA (B) substrates in response to the addition of increasing amounts of TWINKLE as described under “Experimental Procedures.” Changes in anisotropy in the presence of Mg²⁺ (open circles), MgUMPPNP (open triangles) and in the absence of cofactors (filled squares) are shown.

FIGURE 4.

Unwinding kinetics of the 5′- and 3′-tail DNA annealed to M13 ssDNA in the presence of different NTP substrates. The representative 15% acrylamide/TBE gels show the unwinding kinetics of the 5′-tail substrate (A) and 3′-tail substrate (B) at 30 °C in the presence of 25 nm TWINKLE and 4 mm CTP (left panel) and UTP (right panel). Lanes marked ss are the substrate-heated at 95 °C before loading; lanes marked 0 represent time 0. The other lanes in each gel correspond to unwinding reaction times of 3, 7.5, 15, 30, 60, and 90 min. For each NTP, the fraction of DNA unwound at each time point is normalized to the corresponding fraction unwound at time 0, plotted against reaction time, and fitted to Equation 4 for all NTPs except the ones where unwinding was not completed even at 90 min; hence they were fitted to linear Equation 5.

FIGURE 5.

Effect of single strand-binding protein and the reannealing DNA trap on the unwinding kinetics by TWINKLE. A, 5′-tailed substrate was unwound in the presence of 25 nm TWINKLE and 4 mm UTP in the presence of 40 nm unlabeled ssDNA displaced strand trap or 2 μm T7 gp2.5. The rates of unwinding and fraction of unwound ssDNA product with the corresponding S.E. under four different conditions tested in the unwinding assay are summarized in the table. A plot of fraction of DNA unwound over time in the presence of T7 gp2.5 trap (square) and ssDNA trap (triangle), in the absence of trap (circle), and in the presence of T7 gp2.5 trap but absence of TWINKLE (diamond) is also shown. The reaction times are 2, 5, 15, 30, 60, and 90 min. B, unwinding of the longer 55-bp M13ds DNA is better in the presence of DNA trap and gp2.5 than in their absence. The unwinding reaction was carried out under the same conditions as the 20-bp M13ds DNA except that the trap DNA is the 40T55-bp in place of the 40T20-bp DNA. C, plot of fraction of 3′-tailed substrate DNA unwound over time in the presence of 2 μm T7 gp2.5 trap (square), 40 nm ssDNA displaced strand trap (triangle), and in the absence of trap (circle).

FIGURE 6.

DNA strand annealing activity of TWINKLE. A, schematic representation of two partially complementary ssDNAs annealing to form a 40-bp forked DNA. B, annealing assay was performed by mixing 2 nm ssDNA (GC5040tr + GC5040dis, see Table 1) at 30 °C in the presence and in the absence of 50 nm TWINKLE and 4 mm MgUTP for 1, 5, 10, 15, and 30 min. C, 18% acrylamide/TBE gels show the effect of Mg²⁺ and UTP on the annealing of the 40-bp forked DNA at 30 °C in the presence of 10 nm TWINKLE and 2 nm DNA for 0,15, 30, 60, 120, 300, and 600 s. D, amount of annealed product formed is normalized to the amount of product at time 0 and plotted against time and fit to Equation 2 to get the rate and amplitude. The initial annealing rate (fraction annealed/min) in the absence of MgUTP is 0.02 ± 0.002, in the presence of magnesium acetate is 0.03 ± 0.004, in the presence of UTP is 0.01 ± 0.001, and in the presence of MgUTP is 0.04 ± 0.003. E, fraction of annealed product in 2 min at varying TWINKLE hexamer concentrations (6, 12.5, 25, 50, and 100 nm). F, 12% acrylamide/TBE gel shows annealing of 2 nm DNA by 10 nm TWINKLE at 30 °C in 2 min at varying NaCl concentrations (0, 10, 50, 100, 110, 125, 150, 175, 200, 300, 400, and 500 mm). The prequenched controls for each salt concentration are shown in the lanes marked 0 under each salt concentration, and the 2-min reaction for each salt concentration is marked as 2. G, amount of annealed product formed is normalized to the product formed at respective 0 and plotted against salt concentration.

FIGURE 7.

Competition between dsDNA and ssDNA in binding and in TWINKLE-catalyzed strand annealing reaction. A, 12% acrylamide/TBE gels show the annealing of ssDNA to form a 40-bp forked dsDNA in a reaction at 30 °C in the presence of 10 nm TWINKLE, 2 nm DNA, 4 mm UTP, and 6 mm free magnesium acetate conducted for 2 min. In these reactions, TWINKLE was preincubated with increasing concentrations of competitors such as ssDNA (dT100) or dsDNA (17-bp hairpin) for 15 min before adding the ssDNAs to be annealed. B, complex of 6 nm TMR-labeled RD-ds20 and 7 nm TWINKLE was titrated with increasing ssDNAs (5′-TMRtr). The fluorescence anisotropy decrease was fit to Equations 2 and 3 to obtain the ssDNA IC₅₀ of 672 nm and K_i of 174 nm. C, model showing potential ssDNA-binding modes by TWINKLE.

FIGURE 8.

TWINKLE actively recruits ssDNA trap for complementary base-pairing with the displaced strand during unwinding. A, unwinding by TWINKLE was measured in the presence of trap with and without prebound dT100. 25 nm TWINKLE hexamer was prebound to 0.4 nm M1355bpds substrate in the presence of 4 mm UTP before addition of 100 nm dT100. The unwinding reaction was initiated with 6 mm free magnesium and 40 nm displaced strand trap and was quenched after 60 min. The percentage unwound was calculated as before and plotted on a bar chart. Mean percentage unwinding ± S.E., n = 3, Twinkle + Trap = 34.4 ± 5.6, and Twinkle + Trap + dT100 = 7.7 ± 2.9. B, model for displaced strand trap recruitment by TWINKLE to facilitate unwinding.