Steffi Goffart

Twinkle helicase (PEO1) gene mutation causes mitochondrial DNA depletion

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is a clinically and genetically heterogeneous group of autosomal recessive diseases characterized by a reduction in mtDNA copy number. Several nuclear genes have been shown to account for these severe oxidative phosphorylation disorders, but the disease‐causing mutations remain largely unknown.

Regulation and co-ordination of nuclear gene expression during mitochondrial biogenesis.

Biogenesis of mitochondria is happening constantly due to the physiological and developmental situation of a cell. As mitochondrial biogenesis is a complex process producing about 20 % of cellular protein, the expression of the 1000 genes involved is expected to be coordinated and regulated tightly. The variety of physiological stimuli and differentiation states lead to the idea of a complex network connecting many different regulatory pathways. By analysing nuclear encoded mitochondrial genes some of the factors involved in the regulation and coordination of mitochondrial gene expression were identified. These factors include general transcription factors such as Sp1 or YY1, as well as transcription factors specific for mitochondrial genes like the nuclear respiratory factors NRF1 and 2. An important control function linked to the physiological situation of a cell is triggered by hormones such as steroid and thyroid hormones. Even cell type‐specific regulatory proteins like the myogenin transcription factor family have a strong influence on some mitochondrial genes in the specific cellular background. The regulatory function of most of these proteins can be modulated and enhanced by the coactivators PGC‐1α and β and PRC. Although regulatory pathways have been characterized in more detail in recent years, no regulation mechanism has been shown to work on all analysed mitochondrial genes, and the general concept of mitochondrial regulation still remains unclear.

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.

Alterations to the expression level of mitochondrial transcription factor A, TFAM, modify the mode of mitochondrial DNA replication in cultured human cells

Mitochondrial transcription factor A (TFAM) is an abundant mitochondrial protein of the HMG superfamily, with various putative roles in mitochondrial DNA (mtDNA) metabolism. In this study we have investigated the effects on mtDNA replication of manipulating TFAM expression in cultured human cells. Mammalian mtDNA replication intermediates (RIs) fall into two classes, whose mechanistic relationship is not properly understood. One class is characterized by extensive RNA incorporation on the lagging strand, whereas the other has the structure of products of conventional, strand-coupled replication. TFAM overexpression increased the overall abundance of RIs and shifted them substantially towards those of the conventional, strand-coupled type. The shift was most pronounced in the rDNA region and at various replication pause sites and was accompanied by a drop in the relative amount of replication-termination intermediates, a substantial reduction in mitochondrial transcripts, mtDNA decatenation and progressive copy number depletion. TFAM overexpression could be partially phenocopied by treatment of cells with dideoxycytidine, suggesting that its effects are partially attributable to a decreased rate of fork progression. TFAM knockdown also resulted in mtDNA depletion, but RIs remained mainly of the ribosubstituted type, although termination intermediates were enhanced. We propose that TFAM influences the mode of mtDNA replication via its combined effects on different aspects of mtDNA metabolism.

Infantile-onset spinocerebellar ataxia and mitochondrial recessive ataxia syndrome are associated with neuronal complex I defect and mtDNA depletion

Infantile-onset spinocerebellar ataxia (IOSCA) is a severe neurodegenerative disorder caused by the recessive mutation in PEO1, leading to an Y508C change in the mitochondrial helicase Twinkle, in its helicase domain. However, no mitochondrial dysfunction has been found in this disease. We studied here the consequences of IOSCA for the central nervous system, as well as the in vitro performance of the IOSCA mutant protein. The results of the mtDNA analyses were compared to findings in a similar juvenile or adult-onset ataxia syndrome, mitochondrial recessive ataxia syndrome (MIRAS), caused by the W748S mutation in the mitochondrial DNA polymerase (POLG). We show here that IOSCA brain does not harbor mtDNA deletions or increased amount of mtDNA point mutations, whereas MIRAS brain shows multiple deletions of mtDNA. However, IOSCA, and to a lesser extent also MIRAS, show mtDNA depletion in the brain and the liver. In both diseases, especially large neurons show respiratory chain complex I (CI) deficiency, but also CIV is decreased in IOSCA. Helicase activity, hexamerization and nucleoid structure of the IOSCA mutant were, however, unaffected. The lack of in vitro helicase defect or cell culture phenotype suggest that Twinkle-Y508C dysfunction affects mtDNA maintenance in a highly context and cell-type specific manner. Our results indicate that IOSCA is a new member of the mitochondrial DNA depletion syndromes.

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.

Twinkle mutations associated with autosomal dominant progressive external ophthalmoplegia lead to impaired helicase function and in vivo mtDNA replication stalling

Mutations in the mitochondrial helicase Twinkle underlie autosomal dominant progressive external ophthalmoplegia (PEO), as well as recessively inherited infantile-onset spinocerebellar ataxia and rare forms of mitochondrial DNA (mtDNA) depletion syndrome. Familial PEO is typically associated with the occurrence of multiple mtDNA deletions, but the mechanism by which Twinkle dysfunction induces deletion formation has been under debate. Here we looked at the effects of Twinkle adPEO mutations in human cell culture and studied the mtDNA replication in the Deletor mouse model, which expresses a dominant PEO mutation in Twinkle and accumulates multiple mtDNA deletions during life. We show that expression of dominant Twinkle mutations results in the accumulation of mtDNA replication intermediates in cell culture. This indicated severe replication pausing or stalling and caused mtDNA depletion. A strongly enhanced accumulation of replication intermediates was evident also in six-week-old Deletor mice compared with wild-type littermates, even though mtDNA deletions accumulate in a late-onset fashion in this model. In addition, our results in cell culture pointed to a problem of transcription that preceded the mtDNA depletion phenotype and might be of relevance in adPEO pathophysiology. Finally, in vitro assays showed functional defects in the various Twinkle mutants and broadly agreed with the cell culture phenotypes such as the level of mtDNA depletion and the level of accumulation of replication intermediates. On the basis of our results we suggest that mtDNA replication pausing or stalling is the common consequence of Twinkle PEO mutations that predisposes to multiple deletion formation.

Human heart mitochondrial DNA is organized in complex catenated networks containing abundant four-way junctions and replication forks

Analysis of human heart mitochondrial DNA (mtDNA) by electron microscopy and agarose gel electrophoresis revealed a complete absence of the θ-type replication intermediates seen abundantly in mtDNA from all other tissues. Instead only Y- and X-junctional forms were detected after restriction digestion. Uncut heart mtDNA was organized in tangled complexes of up to 20 or more genome equivalents, which could be resolved to genomic monomers, dimers, and linear fragments by treatment with the decatenating enzyme topoisomerase IV plus the cruciform-cutting T7 endonuclease I. Human and mouse brain also contained a population of such mtDNA forms, which were absent, however, from mouse, rabbit, or pig heart. Overexpression in transgenic mice of two proteins involved in mtDNA replication, namely human mitochondrial transcription factor A or the mouse Twinkle DNA helicase, generated abundant four-way junctions in mtDNA of heart, brain, and skeletal muscle. The organization of mtDNA of human heart as well as of mouse and human brain in complex junctional networks replicating via a presumed non-θ mechanism is unprecedented in mammals.

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.

Of circles, forks and humanity: Topological organisation and replication of mammalian mitochondrial DNA.

The organisation of mammalian mitochondrial DNA (mtDNA) is more complex than usually assumed. Despite often being depicted as a simple circle, the topology of mtDNA can vary from supercoiled monomeric circles over catenanes and oligomers to complex multimeric networks. Replication of mtDNA is also not clear cut. Two different mechanisms of replication have been found in cultured cells and in most tissues: a strand‐asynchronous mode involving temporary RNA coverage of one strand, and a strand‐coupled mode rather resembling conventional nuclear DNA replication. In addition, a recombination‐initiated replication mechanism is likely to be associated with the multimeric mtDNA networks found in human heart. Although an insight into the general principles and key factors of mtDNA organisation and maintenance has been gained over the last few years, there are many open questions regarding replication initiation, termination and physiological factors determining mtDNA organisation and replication mode. However, common themes in mtDNA maintenance across eukaryotic kingdoms can provide valuable lessons for future work.