Oxidative DNA damage stalls the human mitochondrial replisome

Oxidative stress is capable of causing damage to various cellular constituents, including DNA. There is however limited knowledge on how oxidative stress influences mitochondrial DNA and its replication. Here, we have used purified mtDNA replication proteins, i.e. DNA polymerase γ holoenzyme, the mitochondrial single-stranded DNA binding protein mtSSB, the replicative helicase Twinkle and the proposed mitochondrial translesion synthesis polymerase PrimPol to study lesion bypass synthesis on oxidative damage-containing DNA templates. Our studies were carried out at dNTP levels representative of those prevailing either in cycling or in non-dividing cells. At dNTP concentrations that mimic those in cycling cells, the replication machinery showed substantial stalling at sites of damage, and these problems were further exacerbated at the lower dNTP concentrations present in resting cells. PrimPol, the translesion synthesis polymerase identified inside mammalian mitochondria, did not promote mtDNA replication fork bypass of the damage. This argues against a conventional role for PrimPol as a mitochondrial translesion synthesis DNA polymerase for oxidative DNA damage; however, we show that Twinkle, the mtDNA replicative helicase, is able to stimulate PrimPol DNA synthesis in vitro, suggestive of an as yet unidentified role of PrimPol in mtDNA metabolism

Molecular insights into mitochondrial DNA replication

Mitochondria are organelles found in eukaryotic cells. These organelles produce most of the adenosine triphosphate that cells use as a source of energy. Mitochondria contain their own genomic material, a circular DNA genome (mtDNA) that encodes subunits of the respiratory chain complexes and RNA components needed for mitochondrial translation. Many aspects of mtDNA replication are still not understood and in this thesis we address some of the molecular mechanisms of this process in mammalian cells. DNA synthesis cannot be initiated de novo, but requires a short RNA primer as a starting point. We here demonstrate that the mitochondrial RNA polymerase (POLRMT) is the primase required for initiation of DNA synthesis from the origin of light strand DNA replication (OriL) in human mtDNA. Using purified POLRMT and the core factors of the mitochondrial replisome, we faithfully reconstitute OriLdependent initiation of replication in vitro. During origin activation, OriL is exposed in its single-stranded conformation and adopts a stem-loop structure. POLRMT initiates primer synthesis from a poly-dT stretch in the single-stranded loop region and after about 25 nt, POLRMT is replaced by the mitochondrial DNA polymerase ! (POL!) and DNA synthesis is initiated. Our findings also suggest that the mitochondrial single-stranded DNA binding protein directs origin-specific initiation by efficiently blocking unspecific initiation events in other regions of the mtDNA genome. To analyze the requirements of OriL in vivo, we have used saturation mutagenesis in the mouse combined with in vitro biochemistry and demonstrated that OriL is essential for mtDNA maintenance. OriL requires a stable stem-loop structure and a pyrimidine-rich sequence in the template strand for proper origin function. The OriL mechanism appears to be conserved, since bioinformatics analyses demonstrated the presence of OriL in the mtDNA of most vertebrates including birds. Our findings suggest that mtDNA replication may be performed by a common mechanism in all vertebrates and lend support to the strand-displacement model for mtDNA replication. A molecular understanding of the mitochondrial DNA replication machinery is also of medical importance. Today, more than 160 mutations in the gene encoding the catalytic subunit of POL! (POL!A) have been associated with human disease. One example is the Y955C mutation, which causes autosomal dominant progressive external ophthalmoplegia, a disorder characterized by the accumulation of multiple mtDNA deletions. The Y955C mutation decreases POL! processivity due to a decreased binding affinity for the incoming deoxyribonucleoside triphosphate. However, it is not clear why this biochemical defect leads to a dominant disease. We have used the reconstituted mammalian mtDNA replisome and studied functional consequences of the dominant Y955C mutation. Our study revealed that the POL!A:Y955C enzyme is prone to stalling at dATP insertion sites and instead enters a polymerase/exonuclease idling mode. The mutant POL!A:Y955C competes with wild-type POL!A for access to the primer template. However, once assembled in the replisome, the wild-type enzyme is no longer affected. Our data therefore provide a mechanism for the mtDNA replication phenotypes seen in patients harboring the Y955C mutation

A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL.

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

ATPase-deficient mitochondrial inner membrane protein ATAD3A disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia

De novo mutations in ATAD3A (ATPase family AAA-domain containing protein 3A) were recently found to cause a neurological syndrome with developmental delay, hypotonia, spasticity, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. Using whole-exome sequencing, we identified a dominantly inherited heterozygous variant c.1064G > A (p.G355D) in ATAD3A in a mother presenting with hereditary spastic paraplegia (HSP) and axonal neuropathy and her son with dyskinetic cerebral palsy, both with disease onset in childhood. HSP is a clinically and genetically heterogeneous disorder of the upper motor neurons. Symptoms beginning in early childhood may resemble spastic cerebral palsy. The function of ATAD3A, a mitochondrial inner membrane AAA ATPase, is yet undefined. AAA ATPases form hexameric rings, which are catalytically dependent on the co-operation of the subunits. The dominant-negative patient mutation affects the Walker A motif, which is responsible for ATP binding in the AAA module of ATAD3A, and we show that the recombinant mutant ATAD3A protein has a markedly reduced ATPase activity. We further show that overexpression of the mutant ATAD3A fragments the mitochondrial network and induces lysosome mass. Similarly, we observed altered dynamics of the mitochondrial network and increased lysosomes in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells. These alterations were verified in patient fibroblasts to associate with upregulated basal autophagy through mTOR inactivation, resembling starvation. Mutations in ATAD3A can thus be dominantly inherited and underlie variable neurological phenotypes, including HSP, with intrafamiliar variability. This finding extends the group of mitochondrial inner membrane AAA proteins associated with spasticity.Peer reviewe

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

Role of endoscopic ultrasonography in the diagnostic work-up of idiopathic acute pancreatitis (PICUS):study protocol for a nationwide prospective cohort study

INTRODUCTION: Idiopathic acute pancreatitis (IAP) remains a dilemma for physicians as it is uncertain whether patients with IAP may actually have an occult aetiology. It is unclear to what extent additional diagnostic modalities such as endoscopic ultrasonography (EUS) are warranted after a first episode of IAP in order to uncover this aetiology. Failure to timely determine treatable aetiologies delays appropriate treatment and might subsequently cause recurrence of acute pancreatitis. Therefore, the aim of the Pancreatitis of Idiopathic origin: Clinical added value of endoscopic UltraSonography (PICUS) Study is to determine the value of routine EUS in determining the aetiology of pancreatitis in patients with a first episode of IAP. METHODS AND ANALYSIS: PICUS is designed as a multicentre prospective cohort study of 106 patients with a first episode of IAP after complete standard diagnostic work-up, in whom a diagnostic EUS will be performed. Standard diagnostic work-up will include a complete personal and family history, laboratory tests including serum alanine aminotransferase, calcium and triglyceride levels and imaging by transabdominal ultrasound, magnetic resonance imaging or magnetic resonance cholangiopancreaticography after clinical recovery from the acute pancreatitis episode. The primary outcome measure is detection of aetiology by EUS. Secondary outcome measures include pancreatitis recurrence rate, severity of recurrent pancreatitis, readmission, additional interventions, complications, length of hospital stay, quality of life, mortality and costs, during a follow-up period of 12 months. ETHICS AND DISSEMINATION: PICUS is conducted according to the Declaration of Helsinki and Guideline for Good Clinical Practice. Five medical ethics review committees assessed PICUS (Medical Ethics Review Committee of Academic Medical Center, University Medical Center Utrecht, Radboud University Medical Center, Erasmus Medical Center and Maastricht University Medical Center). The results will be submitted for publication in an international peer-reviewed journal. TRIAL REGISTRATION NUMBER: Netherlands Trial Registry (NL7066). Prospectively registered

MtDNA Maintenance by Twinkle and Polymerase Gamma in Health and Disease

Most of the energy needed for cellular function is produced by organelles called mitochondria. The total process of this energy production system located within the mitochondria is called oxidative phosphorylation (OXPHOS). Some of the genes that encode essential subunits of this OXPHOS system are located in the mitochondrial DNA (mtDNA), a small circular genome that is separated from the nuclear DNA. For this reason proper maintenance of mtDNA is crucial for life. To understand which factors and mechanisms are involved in mtDNA replication and repair is important since defects in mitochondrial DNA are a common cause of human neuromuscular genetic disease and are also implicated in aging and infertility. The maintenance and replication of mtDNA depends on nuclear encoded genes. A defect in one of the nuclear genes involved in mtDNA maintenance can result in the accumulation of mtDNA abnormalities, like depletion or multiple deletions. Autosomal dominant progressive external ophthalmaplegia (adPEO) with multiple mtDNA deletion is an example of such a disorder. In this disease the multiple mtDNA deletions accumulate progressively during life and cause gradual paralysis of eye movements, ptosis and exercise intolerance all as a consequence of a slow decrease in energy production. Although several distinct autosmal loci for this disorder have been identified, in most cases the underlying nuclear gene defect remains unclear. In this study we tried to identify novel factors involved in mtDNA maintenance and adPEO disease. We sequenced DNA obtained from adPEO patient in order to find the disease-causing nuclear gene defect. Further we measured the in vivo mtDNA replication fidelity in adPEO patient and compared the findings to age matched control. In order to learn more about the adPEO disease mechanism we mapped multiple mtDNA deletions to investigate at what kind of DNA sequences breakpoints occur. Based on this study we proposed a hypothesis of the mechanism behind deletion formation in adPEO. During later studies we used an inducible expression system in human cultured cells to study the functions of two proteins, involved in mtDNA maintenance and associated with adPEO, the novel mitochondrial replication protein Twinkle and POLG1 polymerase. This study created better understanding of the disease process underlying adPEO and the functions of Twinkle and POLG1 in general mtDNA replication. This basic research, discussed in this thesis will contribute to the understanding of human diseases caused by mitochondrial dysfunction that could ultimately result to development of a therapy.Most of the energy needed for cellular function is produced by organelles called mitochondria. The total process of this energy production system located within the mitochondria is called oxidative phosphorylation (OXPHOS). Some of the genes that encode essential subunits of this OXPHOS system are located in the mitochondrial DNA (mtDNA), a small circular genome that is separated from the nuclear DNA. For this reason proper maintenance of mtDNA is crucial for life. To understand which factors and mechanisms are involved in mtDNA replication and repair is important since defects in mitochondrial DNA are a common cause of human neuromuscular genetic disease and are also implicated in aging and infertility. The maintenance and replication of mtDNA depends on nuclear encoded genes. A defect in one of the nuclear genes involved in mtDNA maintenance can result in the accumulation of mtDNA abnormalities, like depletion or multiple deletions. Autosomal dominant progressive external ophthalmaplegia (adPEO) with multiple mtDNA deletion is an example of such a disorder. In this disease the multiple mtDNA deletions accumulate progressively during life and cause gradual paralysis of eye movements, ptosis and exercise intolerance all as a consequence of a slow decrease in energy production. Although several distinct autosmal loci for this disorder have been identified, in most cases the underlying nuclear gene defect remains unclear. In this study we tried to identify novel factors involved in mtDNA maintenance and adPEO disease. We sequenced DNA obtained from adPEO patient in order to find the disease-causing nuclear gene defect. Further we measured the in vivo mtDNA replication fidelity in adPEO patient and compared the findings to age matched control. In order to learn more about the adPEO disease mechanism we mapped multiple mtDNA deletions to investigate at what kind of DNA sequences breakpoints occur. Based on this study we proposed a hypothesis of the mechanism behind deletion formation in adPEO. During later studies we used an inducible expression system in human cultured cells to study the functions of two proteins, involved in mtDNA maintenance and associated with adPEO, the novel mitochondrial replication protein Twinkle and POLG1 polymerase. This study created better understanding of the disease process underlying adPEO and the functions of Twinkle and POLG1 in general mtDNA replication. This basic research, discussed in this thesis will contribute to the understanding of human diseases caused by mitochondrial dysfunction that could ultimately result to development of a therapy