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1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine has been reported to cause parkinsonism via its neurotoxic form, 1-methyl-4-phenylpyridinium ion (MPP + ), which inhibits complex I of the mitochondrial respiratory chain. Its parkinsonism-causing mechanisms attract a great deal of interest as a model of the disease. Recently, we reported that MPP + strongly decreases the amount of mtDNA independent of the inhibition of complex I. Maintenance of a proper amount of mtDNA is essential for the normal function of mitochondria as exemplified in many mitochondrial diseases. The most characteristic feature in vertebral mtDNA replication is that H-strand synthesis proceeds displacing the parental H-strand as a long single strand. It forms the D-loop, a triplex replication intermediate composed of the parental L-strand, nascent H-strand and displaced H-strand. Here we show that MPP + does not inhibit DNA synthesis by DNA polymerase g, but rather releases the nascent H-strands from mtDNA both in organello and in vitro. This indicates that MPP + directly destabilizes the D-loop structure, thereby inhibiting replication. This study raises a new mechanism, i.e. destabilization of replication intermediates, for depletion of mtDNA.

Mammalian mitochondria contain strong nuclease activity. Endonuclease G (endoG), which predominantly resides in mitochondria, accounts for a large part of this nuclease activity. It has been proposed to act as an RNase H-like nuclease on RNAAEDNA hybrids (R-loops) in the D-loop region where the origins of mitochondrial replication are mapped, providing RNA primers for mtDNA replication. However, in contrast with this proposed activity, endoG has recently been shown to translocate to nuclei on apoptotic stimulation and act as a nuclease without sequence specificity. To clarify the role of endoG in mtDNA replication, we examined its submitochondrial localization and its ability to cleave R-loops. At low concentration, it preferentially produces double-stranded breaks in R-loops, but does not act as an RNase H-like nuclease. In addition, it exists in the mitochondrial intermembrane space, but not in the matrix where mtDNA replication occurs. These results do not support the involvement of endoG in mtDNA replication. Based on the fact that guanine tracts, which are preferential targets of endoG, tend to form triplex structures and that endoG produces double-stranded breaks in R-loops, we propose that three-stranded DNA may be the preferred substrate of endoG.Keywords: endonuclease G; mitochondria; mitochondrial DNA; R-loop; triplex DNA.Mammalian mitochondria contain strong nuclease activity which becomes evident when the membranes are disrupted by detergents. Endonuclease G (endoG) accounts for a large part of this mitochondrial nuclease activity. It is essentially a nonspecific nuclease for all nucleic acid species including double-stranded DNA, single-stranded DNA, single-stranded RNA, and RNAAEDNA duplexes [1,2]. As endoG predominantly resides in mitochondria [1], it has been thought to be involved in the metabolism of mtDNA. It is considered that mitochondrial transcripts stably hybridize with template strands around conserved sequence blocks (CSBs) during transcription, forming R-loops consisting of two DNA strands and one RNA strand, and serve as primers for mtDNA replication (Fig. 1A). EndoG cleaves the RNA of a linear RNAAEDNA duplex preferentially in the CSB region [3], raising the possibility that endoG can generate RNA primers for mtDNA replication [3]. However, endoG is not a specific RNase. On the other hand, RNase MRP 1 , which is also thought to provide RNA primers by cleaving the RNA of R-loops, is a specific RNase. In addition, the endogenous RNAAEDNA hybrid is formed in supercoiled mtDNA and should be a triple-stranded R-loop [4]. The cleavage of the RNA of triple-stranded R-loops by endoG has never been shown, while RNase MRP has been shown to cleave the RNA of triple-stranded R-loops preferentially at the CSBs [5]. Furthermore, NUC1, which is a yeast homolog of endoG and is also found in mitochondria, is not essential for mtDNA replication in yeast, as disruption of the gene leads to no obvious derangement of the metabolism of mtDNA [6]. Thus the role of endoG in mtDNA replication is still ambigu...

Ditercalinium chloride was originally synthesized for use as an anticancer drug and was then found to deplete mitochondrial DNA. Ethidium bromide is widely used to deplete mitochondrial DNA and produce mitochondrial DNA-less cell lines. Although ethidium bromide is used in the case of human cell lines, it frequently fails to deplete mitochondrial DNA in mouse cells. In contrast, ditercalinium chloride can deplete mitochondrial DNA in both mouse and human cells. However, little is known of the mechanisms by which ditercalinium chloride depletes mitochondrial DNA. Here, we show that ditercalinium chloride inhibits human DNA polymerase gamma activity as efficiently as does ethidium bromide. Ethidium bromide accumulates much less in mouse B82 cells, as compared with findings in human HeLa cells, whereas ditercalinium chloride accumulates in both to a similar extent. This poor accumulation of ethidium bromide may, in part, account for the resistance. Ethidium bromide distributes diffusely in the mitochondria of HeLa cells, while ditercalinium chloride distributes granularly and hence may be strongly associated with mitochondrial DNA. Each granular spot presumably represents one mitochondrial DNA nucleoid. In support of this idea, ditercalinium chloride co-localizes with Twinkle, a mitochondrial helicase and is assumed to associate with mitochondrial DNA. This close association of ditercalinium chloride with mitochondrial DNA may contribute to the mitochondrial DNA-depleting activity.

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