Twinkle and POLG defects enhance age-dependent accumulation of mutations in the control region of mtDNA.

Autosomal dominant and/or recessive progressive external ophthalmoplegia (ad/arPEO) is associated with mtDNA mutagenesis. It can be caused by mutations in three nuclear genes, encoding the adenine nucleotide translocator 1, the mitochondrial helicase Twinkle or DNA polymerase gamma (POLG). How mutations in these genes result in progressive accumulation of multiple mtDNA deletions in post- mitotic tissues is still unclear.

The mitochondrial theory of aging: dead or alive?

The mitochondrial theory of aging is based around the idea of a vicious cycle, in which somatic mutation of mtDNA engenders respiratory chain dysfunction, enhancing the production of DNA-damaging oxygen radicals. In turn, this is proposed to result in the accumulation of further mtDNA mutations. Finally, a bioenergetic crisis leads to overt tissue dysfunction and degeneration.

Composition and dynamics of human mitochondrial nucleoids.

The organization of multiple mitochondrial DNA (mtDNA) molecules in discrete protein-DNA complexes called nucleoids is well studied in Saccharomyces cerevisiae. Similar structures have recently been observed in human cells by the colocalization of a Twinkle-GFP fusion protein with mtDNA. However, nucleoids in mammalian cells are poorly characterized and are often thought of as relatively simple structures, despite the yeast paradigm.

Capacity of simian immunodeficiency virus strain mac Nef for high-affinity Src homology 3 (SH3) binding revealed by ligand-tailored SH3 domains.

The simian immunodeficiency virus (SIV) Nef protein contains a consensus Src-homology 3 (SH3) binding motif. However, no SH3-domain proteins showing strong binding to SIV Nef have yet been found, and its potential capacity for high-affinity SH3 binding has therefore remained unproven. Here we have used phage-display-assisted protein engineering to develop artificial SH3 domains that bind tightly to SIV strain mac (SIVmac) Nef.

The 7472insC mitochondrial DNA mutation impairs the synthesis and extent of aminoacylation of tRNASer(UCN) but not its structure or rate of turnover.

The 7472insC mitochondrial DNA mutation in the tRNA(Ser(UCN)) gene is associated with sensorineural deafness combined, in some patients, with a wider neurological syndrome. In cultured cybrid cells it causes a 70% decrease in tRNA(Ser(UCN)) abundance and mild respiratory impairment, previously suggested to be due to decreased tRNA stability. When mitochondrial transcription was blocked by ethidium bromide treatment, the half-life of the mutant tRNA was not significantly different from that of wild-type tRNA(Ser(UCN)).

Prominent mitochondrial DNA recombination intermediates in human heart muscle.

Recombination intermediates containing four-way (Holliday) junctions are generated during DNA repair and replication in many systems, including yeast mitochondrial DNA (mtDNA). In contrast, convincing evidence for recombination in mammalian mtDNA is lacking. We have used two-dimensional agarose-gel electrophoresis to analyse non-linear forms of mtDNA in human heart muscle.

Making mitochondrial mutants.

Mitochondrial DNA (mtDNA) encodes a mere 13 polypeptides, all with well-defined cellular functions in mitochondrial energy metabolism. It was first sequenced over two decades ago, yet our understanding of the wider physiological role of mtDNA is surprisingly sketchy. Partly, this reflects the fact that the mitochondrial gene products are essential for life; that is, most mtDNA mutations are expected to be lethal.

The human homologue of the yeast mitochondrial AAA metalloprotease Yme1p complements a yeast yme1 disruptant.

In yeast, three AAA superfamily metalloproteases (Yme1p, Afg3p and Rca1p) are localized to the mitochondrial inner membrane where they perform roles in the assembly and turnover of the respiratory chain complexes. We have investigated the function of the proposed human orthologue of yeast Yme1p, encoded by the YME1L gene on chromosome 10p. Transfection of both HEK-293EBNA and yeast cells with a green fluorescent protein-tagged YME1L cDNA confirmed mitochondrial targeting.

No sex please, we're mitochondria: a hypothesis on the somatic unit of inheritance of mammalian mtDNA.

In this article we develop a model for the organization and maintenance of mitochondrial DNA (mtDNA) in mammalian somatic cells, based on the idea that the unit of genetic function comprises a group of mtDNA molecules that are semi-permanently associated as a mitochondrial nucleoid. Different mtDNA molecules within a nucleoid need not be genetically identical. We propose that nucleoids replicate faithfully via a kind of mitochondrial mitosis, generating daughter nucleoids that are identical copies of each other, but which can themselves segregate freely.

In vivo functional analysis of the human mitochondrial DNA polymerase POLG expressed in cultured human cells.

The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in vivo have not hitherto been documented. By expressing POLG fusion proteins in cultured human cells, we show that the enzyme is targeted to mitochondria, where the Myc epitope-tagged POLG is catalytically active as a DNA polymerase. Long-term culture of cells expressing wild-type POLG-myc revealed no alterations in mitochondrial function.

The np 3243 MELAS mutation: damned if you aminoacylate, damned if you don't.

The np 3243 MELAS mtDNA mutation in tRNA(leu(UUR))has been variously proposed as a loss-of-function or as a gain-of-function mutation, based on apparently contradictory studies in cultured cell lines. A new report describing the molecular effects of the mutation in vivo now mirrors this variability. This should prompt a more systematic re-investigation of cells carrying the mutation, in order to separate primary from secondary and pathogenic from compensatory effects, all of which may contribute to disease phenotype.

Genotypic stability, segregation and selection in heteroplasmic human cell lines containing np 3243 mutant mtDNA.

The mitochondrial genotype of heteroplasmic human cell lines containing the pathological np 3243 mtDNA mutation, plus or minus its suppressor at np 12300, has been followed over long periods in culture. Cell lines containing various different proportions of mutant mtDNA remained generally at a consistent, average heteroplasmy value over at least 30 wk of culture in nonselective media and exhibited minimal mitotic segregation, with a segregation number comparable with mtDNA copy number (>/=1000). Growth in selective medium of cells at 99% np 3243 mutant mtDNA did, however, allow the isolation of clones with lower levels of the mutation, against a background of massive cell death.

Molecular phenotype of the np 7472 deafness-associated mitochondrial mutation in osteosarcoma cell cybrids.

The nucleotide pair (np) 7472 insC mitochondrial DNA mutation in the tRNA(Ser)(UCN) gene is associated with sensorineural deafness, combined in some individuals with a wider syndrome including ataxia and myo-clonus. Previous studies in osteosarcoma cell cybrids revealed only a mild respiratory defect linked to the mutation. We have investigated the biochemical and molecular consequences of the mutation, using a panel of seven osteosarcoma cell cybrids containing 100% mutant mtDNA, plus two cybrids carrying 100% wild-type mtDNA from the same patient.

Mitochondrial tRNALeu isoforms in lung carcinoma cybrid cells containing the np 3243 mtDNA mutation.

We have investigated the representation of structural isoforms of the two mitochondrial leucyl tRNAs in lung carcinoma cybrid cell lines containing the np 3243 (MELAS) mtDNA mutation, alone or in combination with the np 12300 suppressor mutation. The mutant tRNALeu(UUR) is aminoacylated very poorly or not at all, whereas the suppressor tRNALeu(CUN) is efficiently aminoacylated. Deacylated mitochondrial tRNALeu(CUN) is present, in all human cells tested, in two structural isoforms that are separable on denaturing gels, indicating a difference in primary structure.