Takehiro Yasukawa

Mammalian Mitochondrial DNA Replicates Bidirectionally from an Initiation Zone

Previous data from our laboratory suggested that replication of mammalian mitochondrial DNA initiates exclusively at or near to the formerly designated origin of heavy strand replication, OH, and proceeds unidirectionally from that locus. New results obtained using two-dimensional agarose gel electrophoresis of replication intermediates demonstrate that replication of mitochondrial DNA initiates from multiple origins across a broad zone. After fork arrest near OH, replication is restricted to one direction only. The initiation zone of bidirectional replication includes the genes for cytochrome b and NADH dehydrogenase subunits 5 and 6.

Wobble modification defect in tRNA disturbs codon–anticodon interaction in a mitochondrial disease

We previously showed that in mitochondrial tRNALys with an A8344G mutation responsible for myoclonus epilepsy associated with ragged‐red fibers (MERRF), a subgroup of mitochondrial encephalomyopathic diseases, the normally modified wobble base (a 2‐thiouridine derivative) remains unmodified. Since wobble base modifications are essential for translational efficiency and accuracy, we used mitochondrial components to estimate the translational activity in vitro of purified tRNALys carrying the mutation and found no mistranslation of non‐cognate codons by the mutant tRNA, but almost complete loss of translational activity for cognate codons. This defective translation was not explained by a decline in aminoacylation or lowered affinity toward elongation factor Tu. However, when direct interaction of the codon with the mutant tRNALys defective anticodon was examined by ribosomal binding analysis, the wild‐type but not the mutant tRNALys bound to an mRNA–ribosome complex. We therefore concluded that the anticodon base modification defect, which is forced by the pathogenic point mutation, disturbs codon–anticodon pairing in the mutant tRNALys, leading to a severe reduction in mitochondrial translation that eventually could result in the onset of MERRF.

Modification defect at anticodon wobble nucleotide of mitochondrial tRNAs(Leu)(UUR) with pathogenic mutations of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes

The mitochondrial tRNALeu(UUR) (R = A or G) gene possesses several hot spots for pathogenic mutations. A point mutation at nucleotide position 3243 or 3271 is associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes and maternally inherited diabetes with deafness. Detailed studies on two tRNAsLeu(UUR) with the 3243 or 3271 mutation revealed some common characteristics in cybrid cells: (i) a decreased life span, resulting in a 70% decrease in the amounts of the tRNAs in the steady state, (ii) a slight decrease in the ratios of aminoacyl-tRNAsLeu(UUR) versusuncharged tRNAsLeu(UUR), and (iii) accurate aminoacylation with leucine without any misacylation. As a marked result, both of the mutant tRNA molecules were deficient in a modification of uridine that occurs in the normal tRNALeu(UUR) at the first position of the anticodon. The lack of this modification may lead to the mistranslation of leucine into non-cognate phenylalanine codons by mutant tRNAsLeu(UUR), according to the mitochondrial wobble rule, and/or a decrease in the rate of mitochondrial protein synthesis. This finding could explain why two different mutations (3243 and 3271) manifest indistinguishable clinical features.

Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand

Using two‐dimensional agarose gel electrophoresis, we show that mitochondrial DNA (mtDNA) replication of birds and mammals frequently entails ribonucleotide incorporation throughout the lagging strand (RITOLS). Based on a combination of two‐dimensional agarose gel electrophoretic analysis and mapping of 5′ ends of DNA, initiation of RITOLS replication occurs in the major non‐coding region of vertebrate mtDNA and is effectively unidirectional. In some cases, conversion of nascent RNA strands to DNA starts at defined loci, the most prominent of which maps, in mammalian mtDNA, in the vicinity of the site known as the light‐strand origin.

Expression of catalytic mutants of the mtDNA helicase Twinkle and polymerase POLG causes distinct replication stalling phenotypes

The mechanism of mitochondrial DNA replication is a subject of intense debate. One model proposes a strand-asynchronous replication in which both strands of the circular genome are replicated semi-independently while the other model proposes both a bidirectional coupled leading- and lagging-strand synthesis mode and a unidirectional mode in which the lagging-strand is initially laid-down as RNA by an unknown mechanism (RITOLS mode). Both the strand-asynchronous and RITOLS model have in common a delayed synthesis of the DNA-lagging strand. Mitochondrial DNA is replicated by a limited set of proteins including DNA polymerase gamma (POLG) and the helicase Twinkle. Here, we report the effects of expression of various catalytically deficient mutants of POLG1 and Twinkle in human cell culture. Both groups of mutants reduced mitochondrial DNA copy number by severe replication stalling. However, the analysis showed that while induction of POLG1 mutants still displayed delayed lagging-strand synthesis, Twinkle-induced stalling resulted in maturated, essentially fully double-stranded DNA intermediates. In the latter case, limited inhibition of POLG with dideoxycytidine restored the delay between leading- and lagging-strand synthesis. The observed cause-effect relationship suggests that Twinkle-induced stalling increases lagging-strand initiation events and/or maturation mimicking conventional strand-coupled replication.

Defect in modification at the anticodon wobble nucleotide of mitochondrial tRNALys with the MERRF encephalomyopathy pathogenic mutation

A mitochondrial tRNALys gene mutation at nucleotide position 8344 is responsible for the myoclonus epilepsy associated with ragged‐red fibers (MERRF) subgroup of mitochondrial encephalomyopathies. Here, we show that normally modified uridine at the anticodon wobble position remains unmodified in the purified mutant tRNALys. We have reported a similar modification defect at the same position in two mutant mitochondrial tRNAsLeu(UUR) in another subgroup, mitochondrial myopathy, encephalopathy, lactic acidosis and stroke‐like episodes (MELAS), indicating this defect is common in the two kinds of tRNA molecules with the respective mutations of the two major mitochondrial encephalomyopathies. We therefore suggest the defect in the anticodon is responsible, through the translational process, for the pathogenesis of mitochondrial diseases.

Mammalian Mitochondrial DNA Replication Intermediates Are Essentially Duplex but Contain Extensive Tracts of RNA/DNA Hybrid

We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact mitochondrial DNA replication intermediates are essentially duplex throughout their length but contain extensive RNA tracts on one strand. However, the extent of preservation of RNA in such molecules is highly dependent on the preparative method used. These findings strongly support the strand-coupled model of mitochondrial DNA replication involving RNA incorporation throughout the lagging strand.

The accessory subunit of mitochondrial DNA polymerase gamma determines the DNA content of mitochondrial nucleoids in human cultured cells.

The accessory subunit of mitochondrial DNA polymerase γ, POLGβ, functions as a processivity factor in vitro. Here we show POLGβ has additional roles in mitochondrial DNA metabolism. Mitochondrial DNA is arranged in nucleoprotein complexes, or nucleoids, which often contain multiple copies of the mitochondrial genome. Gene-silencing of POLGβ increased nucleoid numbers, whereas over-expression of POLGβ reduced the number and increased the size of mitochondrial nucleoids. Both increased and decreased expression of POLGβ altered nucleoid structure and precipitated a marked decrease in 7S DNA molecules, which form short displacement-loops on mitochondrial DNA. Recombinant POLGβ preferentially bound to plasmids with a short displacement-loop, in contrast to POLGα. These findings support the view that the mitochondrial D-loop acts as a protein recruitment centre, and suggest POLGβ is a key factor in the organization of mitochondrial DNA in multigenomic nucleoprotein complexes.

Wobble modification deficiency in mutant tRNAs in patients with mitochondrial diseases

Point mutations in mitochondrial (mt) tRNA genes are associated with a variety of human mitochondrial diseases. We have shown previously that mt tRNALeu(UUR) with a MELAS A3243G mutation and mt tRNALys with a MERRF A8344G mutation derived from HeLa background cybrid cells are deficient in normal taurine‐containing modifications [τm5(s2)U; 5‐taurinomethyl‐(2‐thio)uridine] at the anticodon wobble position in both cases. The wobble modification deficiency results in defective translation. We report here wobble modification deficiencies of mutant mt tRNAs from cybrid cells with different nuclear backgrounds, as well as from patient tissues. These findings demonstrate the generality of the wobble modification deficiency in mutant tRNAs in MELAS and MERRF.

Mitochondrial single-stranded DNA binding protein is required for maintenance of mitochondrial DNA and 7S DNA but is not required for mitochondrial nucleoid organisation

Single-stranded DNA binding protein (SSB) plays important roles in DNA replication, recombination and repair through binding to single-stranded DNA. The mammalian mitochondrial SSB (mtSSB) is a bacterial type SSB. In vitro, mtSSB was shown to stimulate the activity of the mitochondrial replicative DNA helicase and polymerase, but its in vivo function has not been investigated in detail. Here we studied the role of mtSSB in the maintenance of mitochondrial DNA (mtDNA) in cultured human cells. RNA interference of mtSSB expression in HeLa cells resulted in rapid reduction of the protein and a gradual decline of mtDNA copy number. The rate of mtDNA synthesis showed a moderate decrease upon mtSSB knockdown in HeLa cells. These results confirmed the requirement of mtSSB for mtDNA replication. Many molecules of mammalian mtDNA hold a short third strand, so-called 7S DNA, whose regulation is poorly understood. In contrast to the gradual decrease of mtDNA copy number, 7S DNA was severely reduced upon mtSSB knockdown in HeLa cells. Further, 7S DNA synthesis was significantly affected by mtSSB knockdown in an oseteosarcoma cell line. These data together suggest that mtSSB plays an important role in the maintenance of 7S DNA alongside its role in mtDNA replication. In addition, live-cell staining of mtDNA did not imply alteration in the organisation of mitochondrial nucleoid protein-mtDNA complexes upon mtSSB knockdown in HeLa cells. This result suggests that the presence of 7S DNA is not crucial for the organisation of mitochondrial nucleoids.