SEARCH

SEARCH BY CITATION

Keywords:

  • 1-methyl-4-phenylpyridinium ion;
  • D-loop;
  • Holliday junction;
  • mitochondrial DNA;
  • Parkinson's disease;
  • R-loop

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

During replication, human mitochondrial DNA (mtDNA) takes on a triple-stranded structure known as a D-loop, which is implicated in replication and transcription. 1-Methyl-4-phenylpyridinium ion (MPP+), a toxin inducing parkinsonism, inhibits mtDNA replication, possibly by resolving the D-loops. For initiation of mtDNA replication, mitochondria are thought to have another triple-stranded structure, an R-loop. The R-loop, which is resolved by a bacterial junction-specific helicase, RecG, is also resolved by MPP+. Because mitochondrial D-loops are likewise resolved by RecG, the D- and R-loops may share a similar branched structure. MPP+ resolves cruciform DNA in supercoiled DNA. MPP+ converts a stacked conformation to an extended conformation in a synthetic Holliday junction. This conversion is reversed by 1 mm Mg2+, as is the resolution of the D-loops or cruciform DNA. These observations suggest that the junction structure of mitochondrial D- and R-loops is affected by MPP+.

Abbreviations used
FITC

fluorescein isothiocyanate

mtDNA

mitochondrial DNA

MPP+

1-methyl-4-phenylpyridinium ion.

Parkinson's disease is caused by decrease in dopamine owing to the degeneration of substantia nigra. As 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been reported to cause parkinsonism (Davis et al. 1979; Burns et al. 1983; Langston et al. 1983), it has been extensively studied in an attempt to understand the etiology of Parkinson's disease. MPTP is oxidized to its neurotoxic form, 1-methyl-4-phenylpyridinium ion (MPP+) by monoamine oxidase B in glial cells (Chiba et al. 1984; Singer and Ramsay 1990). Then, MPP+ is accumulated in dopaminergic cells via the dopamine reuptake system (Javitch et al. 1985) and concentrated in mitochondria according to the mitochondrial membrane potential (Ramsay and Singer 1986; Ramsay et al. 1986a,b). MPP+ binds to the same site as rotenone and inhibits NADH-ubiquinone oxidoreductase (complex I) in the mitochondrial respiratory chain (Krueger et al. 1990; Ramsay et al. 1991). The inhibition of complex I by MPP+ in dopaminergic cells is thought to cause MPTP-induced parkinsonism. The decline of mitochondrial respiratory functions are also observed in idiopathic Parkinson's disease (Swerdlow et al. 1996; Schapira 1999).

We have previously reported that MPP+ decreases mitochondrial DNA (mtDNA) in HeLa S3 cells and that the decrease is independent of the inhibition of complex I (Miyako et al. 1997). Human mitochondria have their own DNA that encodes 13 mRNAs for proteins of the respiratory chain, two rRNAs, and 22 tRNAs required in translation. The integrity of mtDNA is essential for normal mitochondrial respiratory functions. A decrease in the copy number of mtDNA is implicated in disease (Arnaudo et al. 1991; Moraes et al. 1991; Poulton et al. 1994). This decrease results in a decline of mitochondrially translated proteins and dysfunction of mitochondria follows. Therefore, sustaining of a certain copy number of mtDNA is critical for maintaining respiratory functions.

mtDNA replication may play a role in regulating the copy number. Mammalian mtDNA acquires a unique triple-stranded replication intermediate structure called the D-loop (Kasamatsu et al. 1971). The D-loop is formed within the non-coding regulatory region (D-loop region) where synthesis of a nascent H-strand proceeds, displacing the parental H-strand. Formation and resolution of D-loops have been implicated in the regulation of mtDNA replication (Clayton 1991). We reported that MPP+ directly destabilizes the mitochondrial D-loop, leading to the inhibition of mtDNA replication and to a subsequent decrease in the amount of mtDNA (Miyako et al. 1997; Miyako et al. 1999; Umeda et al. 2000). These findings indicate that stability of the D-loop is a key element in the regulation of mtDNA replication. mtDNA has another triple-stranded form, the R-loop, which contains single-stranded RNA at mitochondrial conserved sequence blocks (CSB) within the D-loop region. Although its existence has not been directly proven in vivo, the mitochondrial R-loop forms within the CSB region of supercoiled plasmids during in vitro transcription (Xu and Clayton 1996; Ohsato et al. 1999). The mitochondrial R-loop is assumed to provide an RNA primer for mtDNA replication. Therefore, D- and R-loops may have central roles in mechanisms that regulate mtDNA replication.

The involvement of D- and R-loops in DNA replication is not restricted to mtDNA. DNA replication in Escherichia coli can be initiated using homologous intermediary structures (Asai and Kogoma 1994; Kogoma 1996). In E. coli, RecG protein recognizes DNA junctions, including Holliday junctions, and has 3′ to 5′ helicase activity. recG mutants stimulate constitutive stable DNA replication (cSDR). Because cSDR was originally found in Rnase HI-defective mutants, R-loops are thought to be required for the initiation of cSDR (Asai and Kogoma 1994). RecG may remove ectopic R-loops, as does Rnase HI in vivo. RecG resolves R-loops at the ColE1 replication origin, thereby inhibiting the replication of a plasmid containing a ColE1-type replication origin (Fukuoh et al. 1997). RecG also resolves other branched structures, such as Holliday junctions (Lloyd and Sharples 1993), three-way junctions, and triple-stranded junctions (Whitby and Lloyd 1995; McGlynn et al. 1997).

We report that MPP+ resolves mitochondrial D- and R-loops, both of which harbor a triple-stranded branched structure recognized by RecG. MPP+ converts the stacked synthetic four-way junctions into extended junctions. Our observations suggest that DNA branched structures are targets of MPP+.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Reagents and enzymes

MPP+-iodide was purchased from Research Biochemicals Inc. (Natick, MA, USA). Ethidium bromide and chloroquine were from Sigma (St Louis, MO, USA). The restriction enzymes BamHI, EcoRI, and XbaI were from Takara (Seta, Japan) and PstI was from New England Biolabs (Beverly, MA, USA). Other reagents were of analytical grade. Recombinant E. coli RecG protein was purified, as described previously (Fukuoh et al. 1997).

Resolution of R-loops by MPP+

The R-loop was prepared, as described previously (Ohsato et al. 1999), essentially according to Lee and Clayton (1996). In short, the R-loop reaction mixture containing 5 nm pGEMhmD, 50 mm KCl, 20 mm Tris–HCl, pH 8.0, 10 mm MgCl2, 1 mm dithiothreitol (DTT), 0.1 mm NTPs, and 0.2 U/µL SP6 RNA polymerase, was incubated at 37°C for 30 min. The R-loop resolution reaction was carried out in 20 µL of buffer A, containing 40 mm HEPES-KOH, pH 7.5, 25 mm KCl, and the indicated concentration of MPP+ or 6 U of Rnase H, at 37°C for 10 min. The reaction was stopped by adding 1 µg/mL proteinase K and 0.5% sodium dodecylsulfate (SDS), then the preparation was incubated for a further 10 min. The R-loop was analyzed using 0.7% agarose-gel electrophoresis in buffer consisting of 89 mm Tris base, 89 mm boric acid, and 2 mm EDTA. DNA was stained with ethidium bromide.

Detection of D-strands

A crude mitochondrial fraction was prepared from HeLa MRV11 cells by differential centrifugation, as described previously (Kang et al. 1995). Isolated mitochondria were solubilized in 1% SDS, then DNA was extracted using phenol–chloroform. The amount of DNA was determined by measuring the absorbance at 260 nm. The mitochondrial DNA content of extracted DNA was ≈ 10%, as based on Southern blot analysis (results not shown). For D-loop resolution by MPP+, 2 µg of the DNA was incubated in 20 µL of buffer A in the presence of 10 mm MPP+-iodide and the indicated concentration of MgCl2, at 37°C for 10 min. To fully release D-strands, DNA was heated at 90°C for 5 min. A standard reaction for D-loop resolution by RecG was carried out in 20 µL of a mixture containing 3 µg of DNA, 20 mm Tris–HCl, pH 8.0, 50 mm KCl, 4 mm MgCl2, 1 mm DTT, 0.05% bovine serum, 200 nm RecG, and 2 mm ATP, at 37°C for 10 min. The reactions were stopped by adding a loading buffer containing 0.25% bromophenol blue and 80% glycerol. DNA was resolved electrophoretically on a 1.2% agarose gel in buffer (89 mm Tris base, 89 mm boric acid, 2 mm EDTA). For Southern blotting a probe covering the D-loop region (nts 16036–16394) was used. This probe was labeled with alkaline phosphatase using an AlkPhos Direct kit (Amersham Life Science, Buckinghamshire, UK) according to the manufacturer's instructions. The signals were visualized using a chemiluminescent reagent (CDP-Star; Amersham Life Science), recorded using a CCD camera (LAS-1000; Fuji Photo Film, Tokyo, Japan), and then quantified using image analysis software (Image Gauge; Fuji Photo Film).

Cruciform resolution assay

Cruciform formation in the pUC4 plasmid was induced, at 60°C for 2 h, in buffer containing 10 mm HEPES-KOH, pH 7.5, 200 mm KCl (Iwasaki et al. 1991). Two micrograms of pUC4 cruciform DNA was pre-incubated (at 37°C for 15 min) in 20 mm HEPES-KOH, pH 7.5, 25 mm KCl, containing different concentrations of Mg2+, followed by incubation with different concentrations of MPP+ for a further 15 min. Then, 20 U of PstI were added together with a one-tenth volume of 10× H buffer (500 mm Tris–HCl, pH 7.5, 100 mm MgCl2, 10 mm dithiothreitol, 1 m NaCl) followed by incubation for 30 min. After adding loading dye containing 35% Ficoll 400, 0.25% bromophenol blue (wt/vol), and 1% SDS, samples were analyzed using agarose-gel electrophoresis (1.0% gel) in buffer containing 40 mm Tris-acetate, pH 8.0, 1 mm EDTA. DNA was stained with ethidium bromide and visualized under UV light.

Preparation of a four-way junction

A four-way DNA junction was made by annealing four 80-mer oligodeoxynucleotides for junction 3, as described by Duckett et al. (1988). All nucleotides were purchased from Hokkaido System Science (Sapporo, Japan). The sequence of each oligodeoxynucleotide is as follows: 80JCT3B, 5′-CGCAAGCGACAGGAACCTCGAGGGATCCGTCCTAGCA-AGGGCTGCTACCGGAAGCTTCTCGAGGTTCCTGTCGCTTG-CG-3′; 80JCT3H, 5′-CGCAAGCGACAGGAACCTCGAGAAGC-TTCCGGTAGCAGCCTGAGCGGTGGTTGAATTCCTCGAGGT- TCCTGTCGCTTGCG-3′; 80JCT3R, 5′-CGCAAGCGACAGGAA-CCTCGAGGAATTCAACCACCGCTCAACTCAACTGCAGTC-TAGACTCGAGGTTCCTGTCGCTTGCG-3′; 80JCT3X, 5′-CGC-AAGCGACAGGAACCTCGAGTCTAGACTGCAGTTGAGTCC-TTGCTAGGACGGATCCCTCGAGGTTCCTGTCGCTTGCG-3′. The four oligodeoxynucleotides, 0.04 A258 units of each, were annealed in 50 µL of 450 mm NaCl, 24 mm sodium citrate (pH 7.0) at 65°C for 2 h and then allowed to cool slowly (Clegg et al. 1992). For preparation of the fluorescein isothiocianate (FITC)-labeled four-way junction, FITC-labeled 80JCT3B at its 5′ end was used. The four-way junction was recovered by ethanol precipitation and dissolved in 10 mm Tris–HCl, pH 7.5.

The purified junction DNA was digested with appropriate enzymes and analyzed on 5% LongRanger (FMC Bioproducts, Rockland, MA, USA) polyacrylamide gels in buffer (89 mm Tris base, 89 mm boric acid). Other reagents were added both in the gel and buffer system, as indicated. The buffer was continuously recirculated during electrophoresis. The gels were soaked in the buffer for 30 min after electrophoresis and then DNA was visualized using an image scanner (FluorImager 595; Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Unwinding assay

The DNA unwinding capacity of MPP+ was analyzed using a DNA Unwinding Kit (TopoGEN; Columbus, Ohio, USA) according to the manufacturer's instructions. Briefly, supercoiled pBR322 plasmids were treated with 2 IU of DNA-relaxing enzyme human topoisomerase I in buffer (10 mm Tris–HCl, pH 7.5, 1 mm EDTA) for 30 min at 37°C (step I in Fig. 5a). As indicated, EDTA was omitted or replaced with 1 mm MgCl2. The relaxed plasmids were incubated in the presence of MPP+ or ethidium bromide for a further 30 min (step II). SDS (1%) and proteinase K (50 µg/mL) were added to the reaction mixture, which was incubated for 15 min at 56°C and then mixed with an equal volume of chloroform–isoamylalcohol (24 : 1, vol/vol). The resultant aqueous phase was analyzed using agarose-gel electrophoresis (1% gel) in buffer containing 45 mm Tris-phosphate, pH 7.5, 1 mm EDTA. As indicated, 0.4 µm chloroquine was included in the gel and with the buffer. During electrophoresis, a constant voltage of 1 V/cm was applied for 18 h at room temperature (25°C). The gel was stained with 0.5 µg/mL ethidium bromide for 30 min and unbound ethidium bromide was removed by incubation in distilled water for a further 30 min prior to photodocumentation.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Resolution of D-loops

We reported previously that MPP+ resolves mitochondrial D-loops (Umeda et al. 2000). In the present work, the D-loop-resolving activity of MPP+ was shown to decline with increasing concentrations of Mg2+(Fig. 1a). The resolution was inhibited by ≈ 50% at 100 µm Mg2+ (Fig. 1a). The MPP+-induced release of D-strands was almost completely inhibited at 100 mm KCl (Fig. 1b).

image

Figure 1. D-loop resolution by MPP+. (a) mtDNA was incubated with 10 mm MPP+ and the indicated concentration of MgCl2 at 37°C for 60 min. To fully release D-strands, DNA was heated at 90°C for 5 min. The released D-strands were detected using Southern blots. (b) Effects of KCl on the MPP+-induced D-loop resolution.

Download figure to PowerPoint

DNA unwinding by MPP+

Formation of D-loops is dependent on the negative superhelicity of DNA. Reagents that positively unwind DNA reduce negative superhelicity locally (Waring 1981) and can then resolve D-loops (Wiegand et al. 1977). We next measured DNA unwinding by MPP+(Fig. 2a). MPP+ induced supercoiling of relaxed pBR322 plasmids in the presence of topoisomerase I, in a dose-dependent manner (Fig. 2b, upper panel). Because chloroquine gives a positive superhelicity, negatively supercoiled plasmids are thus relaxed. The supercoiled pBR322 was relaxed in the gel containing chloroquine (Fig. 2b, compare the lanes between the upper and lower panels), thereby indicating that negative supercoiling was introduced by topoisomerase I in the presence of MPP+, in other words, that MPP+ positively unwound DNA. A similar pattern was observed with ethidium bromide, which positively unwinds DNA (results not shown).

image

Figure 2. DNA intercalation of MPP+ and ethidium bromide. (a) Scheme used for the unwinding assay. After relaxation of supercoiled pBR322 with topoisomerase I (topo I) (step I), a drug was added, followed by further incubation with topoisomerase I (step II). If a drug binds to DNA and changes superhelicity, topoisomerase I would relax plasmids again by inducing the opposite direction of supercoiling. Thus, after removal of the drug and topoisomerase I, supercoiled plasmids would be observed (step III). On the other hand, if a drug does not bind, plasmids would remain relaxed. (b) Relaxed pBR322 plasmids were incubated with topoisomerase I and various concentrations of MPP+. Then plasmids were electrophoresed in an agarose gel in the absence (upper panel) or presence (lower panel) of 0.4 µm chloroquine. In the latter, 0.4 µm chloroquine was also included in the electrophoresis buffer. (c) Supercoiled pBR322 was first incubated with topoisomerase I (lane 2). The relaxed pBR322 was then incubated with 1 mm MgCl2 (lane 3), 10 mm MPP+ (lane 4), or 0.5 µg/mL ethidium bromide (lane 6). In lane 5, the relaxed pBR322 was incubated with 10 mm MPP+ in the presence of 1 mm MgCl2. In lane 7, supercoiled pBR 322 underwent the first relaxation reaction in the presence of 1 mm MgCl2. R, relaxed form of pBR322; SC, supercoiled pBR322. (d) Effects of ethidium bromide on the D-strand release. mtDNA was incubated with ethidium bromide. MtDNA and the released D-strands were detected using Southern blots.

Download figure to PowerPoint

Mg2+, at a concentration of 1 mm, neither inhibited the relaxation by topoisomerase I nor affected the relaxed plasmids (Fig. 2c, lanes 2, 3, and 7), thus indicating that 1 mm Mg2+ did not affect this unwinding assay system per se. The relaxed plasmids were re-supercoiled by topoisomerase I in the presence of 10 mm MPP+ (Fig. 2c, lane 4). Mg2+, at a concentration of 1 mm, had little effect on the re-supercoiling induced by MPP+ (Fig. 2c, lanes 4 and 5), i.e. DNA unwinding by MPP+. Because 1 mm Mg2+ almost completely inhibited the resolution of mitochondrial D-loops by MPP+ (Fig. 1a), MPP+ may not resolve the D-loops via a non-specific interaction with DNA. Ethidium bromide, at a concentration of 0.5 µg/mL, induced supercoiling more strongly than did 10 mm MPP+ (Fig. 2c, lane 6), whereas ethidium bromide, even at 1 µg/mL, hardly resolved the D-loops (Fig. 2d). Therefore, DNA unwinding is insufficient for D-loop resolution, at least to the extent induced by 10 mm MPP+.

MPP+ resolves R-loops

Mitochondrial R-loops, like D-loops, have a triple-stranded structure that may play an essential role in mtDNA replication. R-loops have been shown to be reconstituted by in vitro transcription using supercoiled circular plasmids (Ohsato et al. 1999). The R-loops migrated more slowly during agarose-gel electrophoresis than the original supercoiled circular plasmids (Fig. 3, lanes 2 and 3). The R-loop formation was confirmed by the reversal of its migration pattern with Rnase H (results not shown). MPP+ resolved R-loops in a dose-dependent manner (Fig. 3, lanes 3–8).

image

Figure 3. Resolution of the R-loops by MPP+. Using supercoiled pGEMhmD plasmids (lane 2, lower arrow), the reconstituted R-loops (lane 3, upper arrow) show a slower migration than seen in the original supercoiled plasmids owing to a more relaxed conformation of R-loops. R-loops were incubated with MPP+ and then analyzed using a 0.7% agarose gel (lanes 3–8). The DNA was stained with ethidium bromide. See lane 1 for molecular-weight markers.

Download figure to PowerPoint

RecG resolves D-loop structure of mtDNA

MPP+ affected the triple-stranded structure irrespective of the third-strand species, DNA or RNA, which means that D- and R-loops share a common structure affected by MPP+. We reported previously that mitochondrial R-loops are resolved by RecG (Ohsato et al. 1999). RecG is an ATP-dependent and branched structure-specific helicase. To identify structures shared by D- and R-loops, we examined the effect of RecG on mitochondrial D-loops. As shown in Fig. 4, RecG also resolved the D-loops and released the third DNA strands, i.e. D-strands, in an ATP-dependent manner, thereby indicating that both D- and R-loops have a RecG-recognizable branched structure.

image

Figure 4. Resolution of the D-loops by RecG. mtDNA was incubated with ATP alone (lane 2), RecG alone (lane 3), or RecG and ATP (lane 4), and subsequently analyzed using Southern blots. For maximal release of the D-strands, the DNA was incubated at 90°C for 5 min (lane 1).

Download figure to PowerPoint

Resolution of cruciform DNA in supercoiled plasmid

We then investigated whether MPP+ could resolve another branched structure, a four-way structure on a plasmid. A cruciform structure was made by taking advantage of the intra-strand base-pairing formation between inverted repeat sequences in the pUC4 plasmid (Iwasaki et al. 1991). The cruciform DNA was sensitive to RecG (results not shown). The intra-strand base-pairing disrupts a unique PstI site in the plasmid (Fig. 5a), while resolution of the cruciform structure restores the PstI site (Fig. 5a). The cruciform DNA was resistant to PstI digestion (Fig. 5b, lanes 1 and 2). MPP+ at 10 mm completely restored the PstI sensitivity of pUC4 (Fig. 5b, lanes 1–10), thereby indicating that the cruciform structure is resolved by MPP+. Prior addition of Mg2+ dose-dependently inhibited the MPP+-induced resolution (Fig. 5c, lanes 3–7). Mg2+, at a concentration of 5 mm, showed almost complete inhibition (compare lane 2 to lane 6 in Fig. 5c). Pre-incubation with 10 mm Mg2+ did not inhibit the digestion of normal pUC4 plasmids by PstI, hence Mg2+ did not affect PstI activity, per se (results not shown).

image

Figure 5. Resolution of the cruciform structure in plasmid PUC4. (a) Schematic representation of disruption of the PstI site by the formation of cruciform DNA. (b) Cruciform DNA was formed in the pUC4 plasmid. Cruciform DNA (2 µg) was incubated with MPP+ followed by digestion with PstI (lanes 2, 4, 6, 8 and 10). L, linearized form; SC, undigested supercoiled circular form. (c) Effects of Mg2+. Cruciform DNA formed in the pUC4 plasmid (lane 1) and was digested by PstI (lane 2). Cruciform DNA was incubated with 10 mm MPP+ in the presence of the indicated concentrations of MgCl2 followed by PstI digestion (lanes 3–7). MWM, molecular-weight marker (lane 8).

Download figure to PowerPoint

To better understand the actions of MPP+, we examined the effects of ethidium bromide. This reagent, a well-known DNA intercalator, has features in common with MPP+: both compounds inhibit mtDNA replication and deplete intracellular mtDNA (King and Attardi 1996; Miyako et al. 1997). Cruciform pUC4 DNA was minimally cleaved, but supercoiled pUC4 was completely cleaved by PstI in the presence of 1 µg/mL ethidium bromide (results not shown), thereby indicating that ethidium bromide does not affect the cruciform. As MPP+ inhibits DNA polymerase gamma only minimally (Umeda et al. 2000) and ethidium bromide did not resolve the D-loops (Fig. 2d), these two reagents may decrease the level of intracellular mtDNA in different ways.

Conformation of a synthetic four-way junction

Our observations suggest that MPP+ alters junction structure. To test this notion, we chose a synthetic four-way DNA junction which can adopt two conformations, stacked and extended. The two conformations are distinguished by mobility shifts in gel electrophoresis (Clegg et al. 1992) (Fig. 6a). The four-way DNA junctions acquired the extended form with 10 µm Mg2+ and the stacked form with 100 µm Mg2+ (Fig. 6b, panels 1 and 2). MPP+ (at a concentration of 10 mm) converted this Mg2+-induced stacked form to the extended one (Fig. 6b, panel 3). The effect of MPP+ was reversed using 1 mm Mg2+ (Fig. 6b, panel 4). Ethidium bromide did not affect the conformation of the synthetic four-way DNA junctions (results not shown).

image

Figure 6. Conformation of the synthetic four-way junctions. (a) Schematic representation of mobility shifts depending on conformation of the four-way junction. The four arms designated B, H, X, and R contain the restriction sites for BamHI, HindIII, XbaI, and EcoRI, respectively. The B arm is labeled with FITC (marked with a circle). Two arms were cleaved and the change in gel mobility was analyzed on 5% polyacrylamide gels. (b) Effects of MPP+ and Mg2+ on the four-way junction were examined. A 2-µmol concentration of the four-way junctions was cleaved with two restriction enzymes. BH, junctions incubated with EcoRI and XbaI; BR, junctions incubated with HindIII and XbaI; BX, junctions with HindIII and EcoRI.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

MPP+ resolved mitochondrial R-loops (Fig. 3) that have a Holliday junction-like branched structure recognized by RecG (Ohsato et al. 1999). The R-loop, a key structure in the replication of human mtDNA (Shadel and Clayton 1997), is considered to create RNA primers for H-strand synthesis (Xu and Clayton 1996). Therefore, the resolution of R-loops, in addition to D-loops, may contribute to the depletion of mtDNA induced by MPP+. The resolution of D-loops by MPP+ was strongly inhibited below a physiological range of KCl, under our in vitro conditions (Fig. 1b). However, D-loops started to resolve within 1 min after the addition of MPP+in organello (Umeda et al. 2000). It seems probable that unknown factors facilitating the resolution in mitochondria are missing in the current in vitro conditions. Some factors may influence the resolution by altering the overall structure of mtDNA. For example, mitochondrial transcription factor A binds to DNA and bends DNA strands (Fisher et al. 1992).

The D-strands have a shorter half-life than mtDNA (Bogenhagen and Clayton 1978). Turnover of D-loops is important for the regulation of mtDNA replication. Net replication rate is related to D-loop formation in human T cells (Kai et al. 1999). Related molecular mechanisms for the turnover are poorly understood. We reported previously that MPP+ releases the D-strands from mitochondrial D-loops without nicking or cleavage of DNA strands (Umeda et al. 2000), thus indicating that MPP+ facilitates spontaneous branch migration of D-strands. One probable mechanism is that MPP+ affects the junction structure of D-loops. In the present study, we found that MPP+ induces the extended conformation in four-way DNA junctions. This extended form facilitates spontaneous branch migration of Holliday junctions (Panyutin et al. 1995). The resolution of mitochondrial D-loops by MPP+ was inhibited by Mg2+ within a range where Mg2+ alters conformation of synthetic four-way junctions. The stacked conformation of Holiday junctions is stabilized under high salt conditions (Clegg et al. 1992). The resolution of D-loops was also inhibited with 100 mm KCl. At present, little is known about junction point structures of D-loops. The junction points of D-loops probably adopt either of two conformations equivalent to stacked and extended forms of Holliday junctions. Based on this hypothesis, D-loops in supercoiled DNA would normally be in a ‘stacked’ (spontaneous branch migration incompetent) form and change into an ‘extended’ (spontaneous branch migration competent) form in the presence of MPP+.

MPP+-induced parkinsonism has been extensively investigated as a model of Parkinson's disease. Studies on MPP+ revealed that MPP+ has pleiotropic effects, e.g. inhibition of complex I (Mizuno et al. 1987), enhancement of oxidative stress (Przedborski et al. 1996; Liberatore et al. 1999), activation of poly (ADP-ribose) polymerase (Mandir et al. 1999), and dopamine oxidation (Lotharius and O'Malley 2000). We propose that DNA with a branched structure may be another target of MPP+.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This research was supported, in part, by Grants-in Aid for Scientific Research from the Ministry of Education, Science, Technology, Sports, and Culture, Japan. We extend special thanks to Drs H. Sumimoto (Kyushu University) and M. Hirano (Columbia University) for critical comments. Language assistance was provided by M. Ohara.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • Arnaudo E., Dalakas M., Shanske S., Moraes C. T., DiMauro S. and Schon E. A. (1991) Depletion of muscle mitochondrial DNA in AIDS patients with zidovudine-induced myopathy. Lancet 337, 508510.
  • Asai T. and Kogoma T. (1994) D-loops and R-loops: alternative mechanisms for the initiation of chromosomal replication in Escherichia coli. J. Bacteriol. 176, 18071812.
  • Bogenhagen D. and Clayton D. A. (1978) Mechanism of mitochondrial DNA replication in mouse 1-cells: kinetics of synthesis and turnover of the initiation sequence. J. Mol. Biol. 119, 4968.
  • Burns R. S., Chiueh C. C., Markey S. P., Ebert M. H., Jacobowitz D. M. and Kopin I. J. (1983) A primate model of parkinsonism: selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Proc. Natl Acad. Sci. USA 80, 45464550.
  • Chiba K., Trevor A. and Castagnoli N. Jr (1984) Metabolism of the neurotoxic tertiary amine, MPTP, by brain monoamine oxidase. Biochem. Biophys. Res. Commun. 120, 574578.
  • Clayton D. A. (1991) Replication and transcription of vertebrate mitochondrial DNA. Annu. Rev. Cell Biol. 7, 453478.
  • Clegg R. M., Murchie A. I. H., Zechel A., Carlberg C., Diekman S. and Lilley D. M. J. (1992) Fluorescence energy transfer analysis of the structure of the four-way DNA junction. Biochemistry 31, 48464856.
  • Davis G. C., Williams A. C., Markey S. P., Ebert M. H., Caine E. D., Reichert C. M. and Kopin I. J. (1979) Chronic Parkinsonism secondary to intravenous injection of meperidine analogues. Psychiatry Res. 1, 249254.
  • Duckett D. R., Murchie A. I. H., Diekmann S., Von Kitzing E., Kemper B. and Lilley D. M. J. (1988) The structure of the Holliday junction, and its resolution. Cell 55, 7989.
  • Fisher R. P., Lisowsky T., Breen G. A. and Clayton D. A. (1992) DNA wrapping and bending by a mitochondrial high mobility group-like transcriptional activator protein. J. Biol. Chem. 267, 33583367.
  • Fukuoh A., Iwasaki H., Ishioka K. and Shinagawa H. (1997) ATP-dependent resolution of R-loops at the ColE1 replication origin by Escherichia coli RecG protein, a Holliday junction-specific helicase. EMBO J. 16, 203209.
  • Iwasaki H., Takahagi M., Shiba T., Nakata A. and Shinagawa H. (1991) Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure. EMBO J. 10, 43814389.
  • Javitch J. A., D'Amato R. J., Strittmatter S. M. and Snyder S. H. (1985) Parkinsonism-inducing neurotoxin, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine: uptake of the metabolite N-methyl-4-phenylpyridine by dopamine neurons explains selective toxicity. Proc. Natl Acad. Sci. USA 82, 21732177.
  • Kai Y., Miyako K., Muta S., Umeda S., Irie T., Hamasaki N., Takeshige K. and Kang D. (1999) Mitochondrial DNA replication in human T lymphocytes is regulated primarily at the H-strand termination site. Biochim. Biophys. Acta 1446, 126134.
  • Kang D., Nishida J., Iyama A., Nakabeppu Y., Furuichi M., Fujiwara T., Sekiguchi M. and Takeshige K. (1995) Intracellular localization of 8-oxo-dGTPase in human cells, with special reference to the role of the enzyme in mitochondria. J. Biol. Chem. 270, 1465914665.
  • Kasamatsu H., Robberson D. L. and Vinograd J. (1971) A novel closed circular mitochondrial DNA with properties of replicating intermediates. Proc. Natl Acad. Sci. USA 68, 22522257.
  • King M. P. and Attardi G. (1996) Isolation of human cell lines lacking mitochondrial DNA. Methods Enzymol. 264, 304313.
  • Kogoma T. (1996) Recombination by replication. Cell 85, 625627.
  • Krueger M. J., Singer T. P., Casida J. E. and Ramsay R. R. (1990) Evidence that the blockade of mitochondrial respiration by the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) involves binding at the same site as the respiratory inhibitor, rotenone. Biochem. Biophys. Res. Commun. 169, 123128.
  • Langston J. W., Ballard P., Tetrud J. W. and Irwin I. (1983) Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis. Science 219, 979980.
  • Lee D. Y. and Clayton D. A. (1996) Properties of primer RNA–DNA hybrid at the mouse mitochondrial DNA leading origin of replication. J. Biol. Chem. 271, 2426224269.
  • Liberatore G. T., Jackson-Lewis V., Vukosavic S., Mandir A. S., Vila M., McAuliffe W. G., Dawson V. L., Dawson T. M. and Przedborski S. (1999) Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease. Nat. Med. 5, 14031409.
  • Lloyd R. G. and Sharples G. L. (1993) Dissociation of synthetic Holliday junctions by E. coli RecG protein. EMBO J. 12, 1722.
  • Lotharius J. and O'Malley K. L. (2000) The parkinsonism-inducing drug 1-methyl-4-phenylpyridinium triggers intracellular dopamine oxidation. A novel mechanism of toxicity. J. Biol. Chem. 275, 3858138588.
  • Mandir A. S., Przedborski S., Jackson-Lewis V., Wang Z. Q., Simbulan-Rosenthal C. M., Smulson M. E., Hoffman B. E., Guastella D. B., Dawson V. L. and Dawson T. M. (1999) Poly (ADP-ribose) polymerase activation mediates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism. Proc. Natl Acad. Sci. USA 96, 57745779.
  • McGlynn P., Al-Deib A. A., Liu J., Marians K. J. and Lloyd R. G. (1997) The DNA replication protein PriA, and the recombination protein RecG, bind D-loops. J. Mol. Biol. 270, 212221.DOI: 10.1006/jmbi.1997.1120
  • Miyako K., Kai Y., Irie T., Takeshige K. and Kang D. (1997) The content of intracellular mitochondrial DNA is decreased by 1-methyl-4-phenylpyridinium ion (MPP+). J. Biol. Chem. 272, 96059608.
  • Miyako K., Irie T., Muta T., Umeda S., Kai Y., Fujiwara T., Takeshige K. and Kang D. (1999) 1-Methyl-4-phenylpyridinium ion (MPP+) selectively inhibits the replication of mitochondrial DNA. Eur. J. Biochem. 259, 412418.
  • Mizuno Y., Sone N. and Saitoh T. (1987) Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenylpyridinium ion on activities of the enzymes in the electron transport system in mouse brain. J. Neurochem. 48, 17871793.
  • Moraes C. T., Shanske S., Tritschler H. J., Aprille J. R., Andreetta F., Bonilla E., Schon E. A. and DiMauro S. (1991) mtDNA depletion with variable tissue expression: a novel genetic abnormality in mitochondrial diseases. Am. J. Hum. Genet. 48, 492501.
  • Ohsato T., Muta T., Fukuoh A., Shinagawa H., Hamasaki N. and Kang D. (1999) R-loop in the replication origin of human mitochondrial DNA is resolved by RecG, a Holliday junction-specific helicase. Biochem. Biophys. Res. Commun. 255, 15.
  • Panyutin I. G., Biswas I. and Hsieh P. (1995) A pivotal role for the structure of the Holliday junction in DNA branch migration. EMBO J. 14, 18191826.
  • Poulton J., Morten K., Freeman-Emmerson C., Potter C., Sewry C., Dubowitz V., Kidd H., Stephenson J., Whitehouse W. and Hansen F. J. (1994) Deficiency of the human mitochondrial transcription factor h-mtTFA in infantile mitochondrial myopathy is associated with mtDNA depletion. Hum. Mol. Genet. 3, 17631769.
  • Przedborski S., Jackson-Lewis V., Yokoyama R., Shibata T., Dawson V. L. and Dawson T. M. (1996) Role of neuronal nitric oxide in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity. Proc. Natl Acad. Sci. USA 93, 45654571.
  • Ramsay R. R. and Singer T. P. (1986) Energy-dependent uptake of N-methyl-4-phenylpyridinium, the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, by mitochondria. J. Biol. Chem. 261, 75857587.
  • Ramsay R. R., Salach J. I., Dadgar J. and Singer T. P. (1986a) Inhibition of mitochondrial NADH dehydrogenase by pyridine derivatives and its possible relation to experimental and idiopathic parkinsonism. Biochem. Biophys. Res. Commun. 135, 269275.
  • Ramsay R. R., Salach J. I. and Singer T. P. (1986b) Uptake of the neurotoxin 1-methyl-4-phenylpyridine (MPP+) by mitochondria and its relation to the inhibition of the mitochondrial oxidation of NAD+-linked substrates by MPP+. Biochem. Biophys. Res. Commun. 134, 743748.
  • Ramsay R. R., Krueger M. J., Youngster S. K., Gluck M. R., Casida J. E. and Singer T. P. (1991) Interaction of 1-methyl-4-phenylpyridinium ion (MPP+) and its analogs with the rotenone/piericidin binding site of NADH dehydrogenase. J. Neurochem. 56, 11841190.
  • Schapira A. H. V. (1999) Mitochondrial involvement in Parkinson's disease, Huntington's disease, hereditary spastic paraplegia and Friedreich's ataxia. Biochim. Biophys. Acta 1410, 159170.
  • Shadel G. S. and Clayton D. A. (1997) Mitochondrial DNA maintenance in vertebrates. Annu. Rev. Biochem. 66, 409435.
  • Singer T. P. and Ramsay R. R. (1990) Mechanism of the neurotoxicity of MPTP. An update. FEBS Lett. 274, 18.
  • Swerdlow R. H., Parks. J. K., Miller S. W., Tuttle J. B., Trimmer P. A., Sheehan J. P., Bennett J. P. J., Davis R. E. and Parker W. D. Jr. (1996) Origin and functional consequences of the complex I defect in Parkinson's disease. Ann. Neurol. 40, 663671.
  • Umeda S., Muta T., Ohsato T., Takamatsu C., Hamasaki N. and Kang D. (2000) The D-loop structure of human mitochondrial DNA is destabilized directly by 1-methyl-4-phenylpyridinium ion (MPP+), a Parkinsonism-causing toxin. Eur. J. Biochem. 267, 200206.
  • Waring M. J. (1981) DNA modification and cancer. Annu. Rev. Biochem. 50, 159192.
  • Whitby M. C. and Lloyd R. G. (1995) Branch migration of three-strand recombination intermediates by RecG, a possible pathway for securing exchanges initiated by 3′-tailed duplex DNA. EMBO J. 14, 33023310.
  • Wiegand R. C., Beattie K. L., Holloman W. K. and Radding C. M. (1977) Uptake of homologous single-stranded fragments by superhelical DNA III: the product and its enzymic conversion to a recombinant molecule. J. Mol. Biol. 116, 805824.
  • Xu B. and Clayton D. A. (1996) RNA–DNA hybrid formation at the human mitochondrial heavy-strand origin ceases at replication start sites: an implication for RNA–DNA hybrids serving as primers. EMBO J. 15, 31353143.