Mitochondrial DNA (mtDNA) deletions are associated with mitochondrial disease, and also accumulate during normal human ageing. The mechanisms underlying mtDNA deletions remain unknown although ...several models have been proposed. Here we use deep sequencing to characterize abundant mtDNA deletions in patients with mutations in mitochondrial DNA replication factors, and show that these have distinct directionality and repeat characteristics. Furthermore, we recreate the deletion formation process in vitro using only purified mitochondrial proteins and defined DNA templates. Based on our in vivo and in vitro findings, we conclude that mtDNA deletion formation involves copy-choice recombination during replication of the mtDNA light strand.
Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these. We ...find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication.
Abstract
The role of Ribonuclease H1 (RNase H1) during primer removal and ligation at the mitochondrial origin of light-strand DNA synthesis (OriL) is a key, yet poorly understood, step in ...mitochondrial DNA maintenance. Here, we reconstitute the replication cycle of L-strand synthesis in vitro using recombinant mitochondrial proteins and model OriL substrates. The process begins with initiation of DNA replication at OriL and ends with primer removal and ligation. We find that RNase H1 partially removes the primer, leaving behind the last one to three ribonucleotides. These 5′-end ribonucleotides disturb ligation, a conclusion which is supported by analysis of RNase H1-deficient patient cells. A second nuclease is therefore required to remove the last ribonucleotides and we demonstrate that Flap endonuclease 1 (FEN1) can execute this function in vitro. Removal of RNA primers at OriL thus depends on a two-nuclease model, which in addition to RNase H1 requires FEN1 or a FEN1-like activity. These findings define the role of RNase H1 at OriL and help to explain the pathogenic consequences of disease causing mutations in RNase H1.
Exome sequencing has recently identified mutations in the gene TANGO2 (transport and Golgi organization 2) as a cause of developmental delay associated with recurrent crises involving rhabdomyolysis, ...cardiac arrhythmias, and metabolic derangements. The disease is not well understood, in part as the cellular function and subcellular localization of the TANGO2 protein remain unknown. Furthermore, the clinical syndrome with its heterogeneity of symptoms, signs, and laboratory findings is still being defined. Here, we describe 11 new cases of TANGO2‐related disease, confirming and further expanding the previously described clinical phenotype. Patients were homozygous or compound heterozygous for previously described exonic deletions or new frameshift, splice site, and missense mutations. All patients showed developmental delay with ataxia, dysarthria, intellectual disability, or signs of spastic diplegia. Of importance, we identify two subjects (aged 12 and 17 years) who have never experienced any overt episode of the catabolism‐induced metabolic crises typical for the disease. Mitochondrial complex II activity was mildly reduced in patients investigated in association with crises but normal in other patients. In one deceased patient, post‐mortem autopsy revealed heterotopic neurons in the cerebral white matter, indicating a possible role for TANGO2 in neuronal migration. Furthermore, we have addressed the subcellular localization of several alternative isoforms of TANGO2, none of which were mitochondrial but instead appeared to have a primarily cytoplasmic localization. Previously described aberrations in Golgi morphology were not observed in cultured skin fibroblasts.
This study aims to investigate the extent to which low job control and heavy physical workload in middle age explain educational differences in all-cause and ischemic heart disease (IHD) mortality ...while accounting for important confounding factors.
The study is based on a register-linked cohort of men who were conscripted into the Swedish military at around the age of 18 in 1969/1970 and were alive and registered in Sweden in 2005 (N=46 565). Cox proportional hazards regression models were built to estimate educational differences in all-cause and IHD mortality and the extent to which this was explained by physical workload and job control around age 55 by calculating the reduction in hazard ratio (HR) after adjustments. Indicators of health, health behavior, and other factors measured during conscription were accounted for.
We found a clear educational gradient for all-cause and IHD mortality (HR 2.07 and 2.47, respectively, for the lowest compared to the highest education level). A substantial part was explained by the differential distribution of the confounding factors. However, work-related factors, especially high physical workload, also played important explanatory roles.
Even after accounting for earlier life factors, low job control and especially high physical workload seem to be important mechanistic factors in explaining educational inequalities in all-cause and IHD mortality. It is therefore important to find ways to reduce physical workload and increase job control in order to decrease inequalities in mortality.
We have analysed 385 mitochondrial and 567 chloroplastic signal sequences of proteins found in the organellar proteomes of
Arabidopsis thaliana. Despite overall similarities, the first 16 residues of ...transit peptides differ remarkably. To test the hypothesis that the N-terminally truncated transit peptides would redirect chloroplastic precursor proteins to mitochondria, we studied import of the N-terminal deletion mutants of ELIP, PetC and Lhcb2.1. The results show that the deletion mutants were neither imported into chloroplasts nor miss-targeted to mitochondria
in vitro and
in vivo, showing that the entire transit peptide is necessary for correct targeting as well as miss-sorting.
Critical perspectives have called for the study of women's entrepreneurship as a route to social change. This 'social turn' claims women are empowered and/or emancipated through entrepreneurship with ...limited problematisation of how these interchangeably used concepts operate. Using an institutional perspective in combination with a narrative approach, we investigate women entrepreneurs' life stories on their 'road to freedom' where entrepreneurial activity enables them to 'break free' from particular gendered constraints. Through juxtaposing women's narratives in the contexts of Saudi Arabia and Sweden, the relationship between empowerment and emancipation is disentangled and (re)conceptualised. The findings distinguish between empowerment narrated as individual practices to achieve freedom for the self within institutional structures and emancipation as narrated as a wish to challenge and change structures of power and reach collative freedom. The yearning for collective emancipation propels women's stories of entrepreneurship by raising expectations for entrepreneurship as a vehicle for institutional change. Such stories may fascinate and inspire others to engage in entrepreneurial endeavours to become empowered, but whether they reach emancipation remains an empirical question to be answered. The performative dimension of entrepreneurial narratives is, however, their ability to turn emancipation into an (un)reachable object of desire, with a quest for even more individual empowerment and entrepreneurial activity, at the same time excluding other forms of human conduct as conducive for change.
There is a group of proteins that are encoded by a single gene, expressed as a single precursor protein and dually targeted to both mitochondria and chloroplasts using an ambiguous targeting peptide. ...Sequence analysis of 43 dual targeted proteins in comparison with 385 mitochondrial proteins and 567 chloroplast proteins ofArabidopsis thaliana revealed an overall significant increase in phenylalanines, leucines, and serines and a decrease in acidic amino acids and glycine in dual targeting peptides （dTPs）. The N-terminal portion of dTPs has significantly more serines than mTPs. The number of arginines is similar to those in mTPs, but almost twice as high as those in cTPs. We have investigated targeting determinants of the dual targeting peptide of Thr-tRNA synthetase （ThrRS-dTP） studying organellar import of N- and C-terminal deletion constructs of ThrRS-dTP coupled to GFR These results show that the 23 amino acid long N-terminal portion of ThrRS-dTP is crucial but not sufficient for the organellar import. The C-terminal deletions revealed that the shortest peptide that was capable of conferring dual targeting was 60 amino acids long. We have purified the ThrRS- dTP（2-60） to homogeneity after its expression as a fusion construct with GST followed by CNBr cleavage and ion exchange chromatography. The purified ThrRS-dTP（2-60） inhibited import of pF1β into mitochondria and of pSSU into chloroplasts at μM concentrations showing that dual and organelle-specific proteins use the same organellar import pathways. Furthermore, the CD spectra of ThrRS-dTP（2-60） indicated that the peptide has the propensity for forming α-helical structure in membrane mimetic environments; however, the membrane charge was not important for the amount of induced helical structure. This is the first study in which a dual targeting peptide has been purified and investigated by biochemical and biophysical means.
The social entrepreneurship (SE) discourse habitually casts social entrepreneurs as heroic, creating economic and social value, whereas those whom they strive to assist are portrayed as disadvantaged ...and in need of interventions. This implies an implicit differentiation between knowing, agentic entrepreneurs and less-knowing, more passive beneficiaries. In this article, we seek to unfold the subtle ordering of relations in SE and problematize power-related aspects and ideological influences that potentially overshadow dimensions of mutuality and relationality inherent to SE. From an examination of two cases, we hypothesize that differentiations between knowing selves and learning others tend to disintegrate when entrepreneurs and beneficiaries enter into closer interaction. Adopting a postcolonial framework, we identify three forms of relations in SE: transactional, ambiguous/interactional and transcending.
•Social entrepreneurship exhibits a hierarchization between superior entrepreneurs and beneficiaries in need of intervention.•These power related differentiations overshadow dimensions of mutuality, also inherent to social entrepreneurship.•Examining two cases, we argue that differentiations may begin to disintegrate when interactions are personalized.•Relations may then transform from transactional to ambiguous/interactional.•Leaning on previous critical research we hypothesize a third type of relation in which relations become more mutual.•We conclude that relations between entrepreneurs and beneficiaries take different forms and need to be better understood.
Purpose
Following the example of the critical management education tradition, the purpose of this paper is to argue whether we should keep EE vital by disturbing it, in particular by interrogating ...that which has seemingly become “untouchable” from interrogation.
Design/methodology/approach
This paper takes inspiration from Paolo Freire’s work by proposing a pedagogical approach to entrepreneurship education which builds on an iterative and interactive process, oscillating between deconstructing and reconstructing entrepreneurship, creating space for invention in the classroom. The paper provides exemplary contributions in developing suggestions as to ways forward.
Findings
The ways forward being proposed in this paper include entrepreneurship educators engaging students as co-learners, and evoking their curiosity to pose new questions about the phenomenon; “grounding” students in their own creativity and supporting them to build the confidence needed to develop alternative understandings of how entrepreneurship can function – for themselves, in their future organizations and for society as a whole; and challenging our own teaching positions, and adopting a pedagogical process of invention, stimulating curiosity, co-creation, thought-provoking questions and entrepreneurial action.
Originality/value
This paper provides ways forward in keeping EE “fresh”, by sketching how we need to teach about entrepreneurship, adopting the critical insights emerging in the field. The paper argues how we do not only need other models and approaches to understand entrepreneurship, but also to understand learning and education.
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