Enzymatic removal of ribonucleotides from DNA is essential for mammalian genome integrity and development.

The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells.

Elevated cholesterol in ATAD3 mutants is a compensatory mechanism that leads to membrane cholesterol aggregation.

Aberrant cholesterol metabolism causes neurological disease and neurodegeneration, and mitochondria have been linked to perturbed cholesterol homeostasis via the study of pathological mutations in the ATAD3 gene cluster. However, whether the cholesterol changes were compensatory or contributory to the disorder was unclear, and the effects on cell membranes and the wider cell were also unknown. Using patient-derived cells, we show that cholesterol perturbation is a conserved feature of pathological ATAD3 variants that is accompanied by an expanded lysosome population containing membrane whorls characteristic of lysosomal storage diseases. Lysosomes are also more numerous in Drosophila neural progenitor cells expressing mutant Atad3, which exhibit abundant membrane-bound cholesterol aggregates, many of which co-localize with lysosomes. By subjecting the Drosophila Atad3 mutant to nutrient restriction and cholesterol supplementation, we show that the mutant displays heightened cholesterol dependence. Collectively, these findings suggest that elevated cholesterol enhances tolerance to pathological ATAD3 variants; however, this comes at the cost of inducing cholesterol aggregation in membranes, which lysosomal clearance only partly mitigates.

Minimizing the damage: repair pathways keep mitochondrial DNA intact.

Mitochondrial DNA (mtDNA) faces the universal challenges of genome maintenance: the accurate replication, transmission and preservation of its integrity throughout the life of the organism. Although mtDNA was originally thought to lack DNA repair activity, four decades of research on mitochondria have revealed multiple mtDNA repair pathways, including base excision repair, single-strand break repair, mismatch repair and possibly homologous recombination. These mtDNA repair pathways are mediated by enzymes that are similar in activity to those operating in the nucleus, and in all cases identified so far in mammals, they are encoded by nuclear genes.

Recurrent De Novo NAHR Reciprocal Duplications in the ATAD3 Gene Cluster Cause a Neurogenetic Trait with Perturbed Cholesterol and Mitochondrial Metabolism.

Recent studies have identified both recessive and dominant forms of mitochondrial disease that result from ATAD3A variants. The recessive form includes subjects with biallelic deletions mediated by non-allelic homologous recombination. We report five unrelated neonates with a lethal metabolic disorder characterized by cardiomyopathy, corneal opacities, encephalopathy, hypotonia, and seizures in whom a monoallelic reciprocal duplication at the ATAD3 locus was identified. Analysis of the breakpoint junction fragment indicated that these 67 kb heterozygous duplications were likely mediated by non-allelic homologous recombination at regions of high sequence identity in ATAD3A exon 11 and ATAD3C exon 7. At the recombinant junction, the duplication allele produces a fusion gene derived from ATAD3A and ATAD3C, the protein product of which lacks key functional residues. Analysis of fibroblasts derived from two affected individuals shows that the fusion gene product is expressed and stable. These cells display perturbed cholesterol and mitochondrial DNA organization similar to that observed for individuals with severe ATAD3A deficiency. We hypothesize that the fusion protein acts through a dominant-negative mechanism to cause this fatal mitochondrial disorder. Our data delineate a molecular diagnosis for this disorder, extend the clinical spectrum associated with structural variation at the ATAD3 locus, and identify a third mutational mechanism for ATAD3 gene cluster variants. These results further affirm structural variant mutagenesis mechanisms in sporadic disease traits, emphasize the importance of copy number analysis in molecular genomic diagnosis, and highlight some of the challenges of detecting and interpreting clinically relevant rare gene rearrangements from next-generation sequencing data.

MRPS25 mutations impair mitochondrial translation and cause encephalomyopathy.

Mitochondrial disorders are clinically and genetically heterogeneous and are associated with a variety of disease mechanisms. Defects of mitochondrial protein synthesis account for the largest subgroup of disorders manifesting with impaired respiratory chain capacity; yet, only a few have been linked to dysfunction in the protein components of the mitochondrial ribosomes. Here, we report a subject presenting with dyskinetic cerebral palsy and partial agenesis of the corpus callosum, while histochemical and biochemical analyses of skeletal muscle revealed signs of mitochondrial myopathy. Using exome sequencing, we identified a homozygous variant c.215C>T in MRPS25, which encodes for a structural component of the 28S small subunit of the mitochondrial ribosome (mS25). The variant segregated with the disease and substitutes a highly conserved proline residue with leucine (p.P72L) that, based on the high-resolution structure of the 28S ribosome, is predicted to compromise inter-protein contacts and destabilize the small subunit. Concordant with the in silico analysis, patient's fibroblasts showed decreased levels of MRPS25 and other components of the 28S subunit. Moreover, assembled 28S subunits were scarce in the fibroblasts with mutant mS25 leading to impaired mitochondrial translation and decreased levels of multiple respiratory chain subunits. Crucially, these abnormalities were rescued by transgenic expression of wild-type MRPS25 in the mutant fibroblasts. Collectively, our data demonstrate the pathogenicity of the p.P72L variant and identify MRPS25 mutations as a new cause of mitochondrial translation defect.

Uniparental isodisomy of chromosome 2 causing MRPL44-related multisystem mitochondrial disease.

Mutations in nuclear-encoded protein subunits of the mitochondrial ribosome are an increasingly recognised cause of oxidative phosphorylation system (OXPHOS) disorders. Among them, mutations in the MRPL44 gene, encoding a structural protein of the large subunit of the mitochondrial ribosome, have been identified in four patients with OXPHOS defects and early-onset hypertrophic cardiomyopathy with or without additional clinical features. A 23-year-old individual with cardiac and skeletal myopathy, neurological involvement, and combined deficiency of OXPHOS complexes in skeletal muscle was clinically and genetically investigated. Analysis of whole-exome sequencing data revealed a homozygous mutation in MRPL44 (c.467 T > G), which was not present in the biological father, and a region of homozygosity involving most of chromosome 2, raising the possibility of uniparental disomy. Short-tandem repeat and genome-wide SNP microarray analyses of the family trio confirmed complete maternal uniparental isodisomy of chromosome 2. Mitochondrial ribosome assembly and mitochondrial translation were assessed in patient derived-fibroblasts. These studies confirmed that c.467 T > G affects the stability or assembly of the large subunit of the mitochondrial ribosome, leading to impaired mitochondrial protein synthesis and decreased levels of multiple OXPHOS components. This study provides evidence of complete maternal uniparental isodisomy of chromosome 2 in a patient with MRPL44-related disease, and confirms that MRLP44 mutations cause a mitochondrial translation defect that may present as a multisystem disorder with neurological involvement.

PrimPol, an archaic primase/polymerase operating in human cells.

We describe a second primase in human cells, PrimPol, which has the ability to start DNA chains with deoxynucleotides unlike regular primases, which use exclusively ribonucleotides. Moreover, PrimPol is also a DNA polymerase tailored to bypass the most common oxidative lesions in DNA, such as abasic sites and 8-oxoguanine. Subcellular fractionation and immunodetection studies indicated that PrimPol is present in both nuclear and mitochondrial DNA compartments. PrimPol activity is detectable in mitochondrial lysates from human and mouse cells but is absent from mitochondria derived from PRIMPOL knockout mice. PRIMPOL gene silencing or ablation in human and mouse cells impaired mitochondrial DNA replication. On the basis of the synergy observed with replicative DNA polymerases Polγ and Polε, PrimPol is proposed to facilitate replication fork progression by acting as a translesion DNA polymerase or as a specific DNA primase reinitiating downstream of lesions that block synthesis during both mitochondrial and nuclear DNA replication.

The mitochondrial R-loop.

The DNA in mitochondria contributes essential components of the organelle's energy producing machinery that is essential for life. In 1971, many mitochondrial DNA molecules were found to have a third strand of DNA that maps to a region containing critical regulatory elements for transcription and replication. Forty-five years later, a third strand of RNA in the same region has been reported. This mitochondrial R-loop is present on thousands of copies of mitochondrial DNA per cell making it potentially the most abundant R-loop in nature. Here, I assess the discovery of the mitochondrial R-loop, discuss why it remained unrecognized for almost half a century and propose for it central roles in the replication, organization and expression of mitochondrial DNA, which if compromised can lead to disease states.

Transcript availability dictates the balance between strand-asynchronous and strand-coupled mitochondrial DNA replication.

Mammalian mitochondria operate multiple mechanisms of DNA replication. In many cells and tissues a strand-asynchronous mechanism predominates over coupled leading and lagging-strand DNA synthesis. However, little is known of the factors that control or influence the different mechanisms of replication, and the idea that strand-asynchronous replication entails transient incorporation of transcripts (aka bootlaces) is controversial. A firm prediction of the bootlace model is that it depends on mitochondrial transcripts. Here, we show that elevated expression of Twinkle DNA helicase in human mitochondria induces bidirectional, coupled leading and lagging-strand DNA synthesis, at the expense of strand-asynchronous replication; and this switch is accompanied by decreases in the steady-state level of some mitochondrial transcripts. However, in the so-called minor arc of mitochondrial DNA where transcript levels remain high, the strand-asynchronous replication mechanism is instated. Hence, replication switches to a strand-coupled mechanism only where transcripts are scarce, thereby establishing a direct correlation between transcript availability and the mechanism of replication. Thus, these findings support a critical role of mitochondrial transcripts in the strand-asynchronous mechanism of mitochondrial DNA replication; and, as a corollary, mitochondrial RNA availability and RNA/DNA hybrid formation offer means of regulating the mechanisms of DNA replication in the organelle.

Aberrant ribonucleotide incorporation and multiple deletions in mitochondrial DNA of the murine MPV17 disease model.

All DNA polymerases misincorporate ribonucleotides despite their preference for deoxyribonucleotides, and analysis of cultured cells indicates that mammalian mitochondrial DNA (mtDNA) tolerates such replication errors. However, it is not clear to what extent misincorporation occurs in tissues, or whether this plays a role in human disease. Here, we show that mtDNA of solid tissues contains many more embedded ribonucleotides than that of cultured cells, consistent with the high ratio of ribonucleotide to deoxynucleotide triphosphates in tissues, and that riboadenosines account for three-quarters of them. The pattern of embedded ribonucleotides changes in a mouse model of Mpv17 deficiency, which displays a marked increase in rGMPs in mtDNA. However, while the mitochondrial dGTP is low in the Mpv17-/- liver, the brain shows no change in the overall dGTP pool, leading us to suggest that Mpv17 determines the local concentration or quality of dGTP. Embedded rGMPs are expected to distort the mtDNA and impede its replication, and elevated rGMP incorporation is associated with early-onset mtDNA depletion in liver and late-onset multiple deletions in brain of Mpv17-/- mice. These findings suggest aberrant ribonucleotide incorporation is a primary mtDNA abnormality that can result in pathology.

Pathological ribonuclease H1 causes R-loop depletion and aberrant DNA segregation in mitochondria.

The genetic information in mammalian mitochondrial DNA is densely packed; there are no introns and only one sizeable noncoding, or control, region containing key cis-elements for its replication and expression. Many molecules of mitochondrial DNA bear a third strand of DNA, known as "7S DNA," which forms a displacement (D-) loop in the control region. Here we show that many other molecules contain RNA as a third strand. The RNA of these R-loops maps to the control region of the mitochondrial DNA and is complementary to 7S DNA. Ribonuclease H1 is essential for mitochondrial DNA replication; it degrades RNA hybridized to DNA, so the R-loop is a potential substrate. In cells with a pathological variant of ribonuclease H1 associated with mitochondrial disease, R-loops are of low abundance, and there is mitochondrial DNA aggregation. These findings implicate ribonuclease H1 and RNA in the physical segregation of mitochondrial DNA, perturbation of which represents a previously unidentified disease mechanism.

MPV17 Loss Causes Deoxynucleotide Insufficiency and Slow DNA Replication in Mitochondria.

MPV17 is a mitochondrial inner membrane protein whose dysfunction causes mitochondrial DNA abnormalities and disease by an unknown mechanism. Perturbations of deoxynucleoside triphosphate (dNTP) pools are a recognized cause of mitochondrial genomic instability; therefore, we determined DNA copy number and dNTP levels in mitochondria of two models of MPV17 deficiency. In Mpv17 ablated mice, liver mitochondria showed substantial decreases in the levels of dGTP and dTTP and severe mitochondrial DNA depletion, whereas the dNTP pool was not significantly altered in kidney and brain mitochondria that had near normal levels of DNA. The shortage of mitochondrial dNTPs in Mpv17-/- liver slows the DNA replication in the organelle, as evidenced by the elevated level of replication intermediates. Quiescent fibroblasts of MPV17-mutant patients recapitulate key features of the primary affected tissue of the Mpv17-/- mice, displaying virtual absence of the protein, decreased dNTP levels and mitochondrial DNA depletion. Notably, the mitochondrial DNA loss in the patients' quiescent fibroblasts was prevented and rescued by deoxynucleoside supplementation. Thus, our study establishes dNTP insufficiency in the mitochondria as the cause of mitochondrial DNA depletion in MPV17 deficiency, and identifies deoxynucleoside supplementation as a potential therapeutic strategy for MPV17-related disease. Moreover, changes in the expression of factors involved in mitochondrial deoxynucleotide homeostasis indicate a remodeling of nucleotide metabolism in MPV17 disease models, which suggests mitochondria lacking functional MPV17 have a restricted purine mitochondrial salvage pathway.

ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism.

Although mitochondrial disorders are clinically heterogeneous, they frequently involve the central nervous system and are among the most common neurogenetic disorders. Identifying the causal genes has benefited enormously from advances in high-throughput sequencing technologies; however, once the defect is known, researchers face the challenge of deciphering the underlying disease mechanism. Here we characterize large biallelic deletions in the region encoding the ATAD3C, ATAD3B and ATAD3A genes. Although high homology complicates genomic analysis of the ATAD3 defects, they can be identified by targeted analysis of standard single nucleotide polymorphism array and whole exome sequencing data. We report deletions that generate chimeric ATAD3B/ATAD3A fusion genes in individuals from four unrelated families with fatal congenital pontocerebellar hypoplasia, whereas a case with genomic rearrangements affecting the ATAD3C/ATAD3B genes on one allele and ATAD3B/ATAD3A genes on the other displays later-onset encephalopathy with cerebellar atrophy, ataxia and dystonia. Fibroblasts from affected individuals display mitochondrial DNA abnormalities, associated with multiple indicators of altered cholesterol metabolism. Moreover, drug-induced perturbations of cholesterol homeostasis cause mitochondrial DNA disorganization in control cells, while mitochondrial DNA aggregation in the genetic cholesterol trafficking disorder Niemann-Pick type C disease further corroborates the interdependence of mitochondrial DNA organization and cholesterol. These data demonstrate the integration of mitochondria in cellular cholesterol homeostasis, in which ATAD3 plays a critical role. The dual problem of perturbed cholesterol metabolism and mitochondrial dysfunction could be widespread in neurological and neurodegenerative diseases.

Mitochondrial RNA polymerase is needed for activation of the origin of light-strand DNA replication.

Mitochondrial DNA is replicated by a unique enzymatic machinery, which is distinct from the replication apparatus used for copying the nuclear genome. We examine here the mechanisms of origin-specific initiation of lagging-strand DNA synthesis in human mitochondria. We demonstrate that the mitochondrial RNA polymerase (POLRMT) is the primase required for initiation of DNA synthesis from the light-strand origin of DNA replication (OriL). Using only purified POLRMT and DNA replication factors, we can faithfully reconstitute OriL-dependent initiation in vitro. Leading-strand DNA synthesis is initiated from the heavy-strand origin of DNA replication and passes OriL. The single-stranded OriL is exposed and adopts a stem-loop structure. At this stage, POLRMT initiates primer synthesis from a poly-dT stretch in the single-stranded loop region. After about 25 nt, POLRMT is replaced by DNA polymerase gamma, and DNA synthesis commences. Our findings demonstrate that POLRMT can function as an origin-specific primase in mammalian mitochondria.

Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication.

Encoding ribonuclease H1 (RNase H1) degrades RNA hybridized to DNA, and its function is essential for mitochondrial DNA maintenance in the developing mouse. Here we define the role of RNase H1 in mitochondrial DNA replication. Analysis of replicating mitochondrial DNA in embryonic fibroblasts lacking RNase H1 reveals retention of three primers in the major noncoding region (NCR) and one at the prominent lagging-strand initiation site termed Ori-L. Primer retention does not lead immediately to depletion, as the persistent RNA is fully incorporated in mitochondrial DNA. However, the retained primers present an obstacle to the mitochondrial DNA polymerase γ in subsequent rounds of replication and lead to the catastrophic generation of a double-strand break at the origin when the resulting gapped molecules are copied. Hence, the essential role of RNase H1 in mitochondrial DNA replication is the removal of primers at the origin of replication.

A robust assay to measure DNA topology-dependent protein binding affinity.

DNA structure and topology pervasively influence aspects of DNA metabolism including replication, transcription and segregation. However, the effects of DNA topology on DNA-protein interactions have not been systematically explored due to limitations of standard affinity assays. We developed a method to measure protein binding affinity dependence on the topology (topological linking number) of supercoiled DNA. A defined range of DNA topoisomers at equilibrium with a DNA binding protein is separated into free and protein-bound DNA populations using standard nitrocellulose filter binding techniques. Electrophoretic separation and quantification of bound and free topoisomers combined with a simple normalization procedure provide the relative affinity of the protein for the DNA as a function of linking number. Employing this assay we measured topology-dependent DNA binding of a helicase, a type IB topoisomerase, a type IIA topoisomerase, a non-specific mitochondrial DNA binding protein and a type II restriction endonuclease. Most of the proteins preferentially bind negatively supercoiled DNA but the details of the topology-dependent affinity differ among proteins in ways that expose differences in their interactions with DNA. The topology-dependent binding assay provides a robust and easily implemented method to probe topological influences on DNA-protein interactions for a wide range of DNA binding proteins.

Unique features of DNA replication in mitochondria: a functional and evolutionary perspective.

Last year, we reported a new mechanism of DNA replication in mammals. It occurs inside mitochondria and entails the use of processed transcripts, termed bootlaces, which hybridize with the displaced parental strand as the replication fork advances. Here we discuss possible reasons why such an unusual mechanism of DNA replication might have evolved. The bootlace mechanism can minimize the occurrence and impact of single-strand breaks that would otherwise threaten genome stability. Furthermore, by providing an implicit mismatch recognition system, it should limit the occurrence of replication-dependent deletions and insertions, and defend against invading elements. Such a mechanism may also limit attempts to manipulate the mammalian mitochondrial genome.

MPV17L2 is required for ribosome assembly in mitochondria.

MPV17 is a mitochondrial protein of unknown function, and mutations in MPV17 are associated with mitochondrial deoxyribonucleic acid (DNA) maintenance disorders. Here we investigated its most similar relative, MPV17L2, which is also annotated as a mitochondrial protein. Mitochondrial fractionation analyses demonstrate MPV17L2 is an integral inner membrane protein, like MPV17. However, unlike MPV17, MPV17L2 is dependent on mitochondrial DNA, as it is absent from ρ(0) cells, and co-sediments on sucrose gradients with the large subunit of the mitochondrial ribosome and the monosome. Gene silencing of MPV17L2 results in marked decreases in the monosome and both subunits of the mitochondrial ribosome, leading to impaired protein synthesis in the mitochondria. Depletion of MPV17L2 also induces mitochondrial DNA aggregation. The DNA and ribosome phenotypes are linked, as in the absence of MPV17L2 proteins of the small subunit of the mitochondrial ribosome are trapped in the enlarged nucleoids, in contrast to a component of the large subunit. These findings suggest MPV17L2 contributes to the biogenesis of the mitochondrial ribosome, uniting the two subunits to create the translationally competent monosome, and provide evidence that assembly of the small subunit of the mitochondrial ribosome occurs at the nucleoid.

Mitochondrial transcription terminator family members mTTF and mTerf5 have opposing roles in coordination of mtDNA synthesis.

All genomes require a system for avoidance or handling of collisions between the machineries of DNA replication and transcription. We have investigated the roles in this process of the mTERF (mitochondrial transcription termination factor) family members mTTF and mTerf5 in Drosophila melanogaster. The two mTTF binding sites in Drosophila mtDNA, which also bind mTerf5, were found to coincide with major sites of replication pausing. RNAi-mediated knockdown of either factor resulted in mtDNA depletion and developmental arrest. mTTF knockdown decreased site-specific replication pausing, but led to an increase in replication stalling and fork regression in broad zones around each mTTF binding site. Lagging-strand DNA synthesis was impaired, with extended RNA/DNA hybrid segments seen in replication intermediates. This was accompanied by the accumulation of recombination intermediates and nicked/broken mtDNA species. Conversely, mTerf5 knockdown led to enhanced replication pausing at mTTF binding sites, a decrease in fragile replication intermediates containing single-stranded segments, and the disappearance of species containing segments of RNA/DNA hybrid. These findings indicate an essential and previously undescribed role for proteins of the mTERF family in the integration of transcription and DNA replication, preventing unregulated collisions and facilitating productive interactions between the two machineries that are inferred to be essential for completion of lagging-strand DNA synthesis.

Mitochondrial DNA replication proceeds via a 'bootlace' mechanism involving the incorporation of processed transcripts.

The observation that long tracts of RNA are associated with replicating molecules of mitochondrial DNA (mtDNA) suggests that the mitochondrial genome of mammals is copied by an unorthodox mechanism. Here we show that these RNA-containing species are present in living cells and tissue, based on interstrand cross-linking. Using DNA synthesis in organello, we demonstrate that isolated mitochondria incorporate radiolabeled RNA precursors, as well as DNA precursors, into replicating DNA molecules. RNA-containing replication intermediates are chased into mature mtDNA, to which they are thus in precursor-product relationship. While a DNA chain terminator rapidly blocks the labeling of mitochondrial replication intermediates, an RNA chain terminator does not. Furthermore, processed L-strand transcripts can be recovered from gel-extracted mtDNA replication intermediates. Therefore, instead of concurrent DNA and RNA synthesis, respectively, on the leading and lagging strands, preformed processed RNA is incorporated as a provisional lagging strand during mtDNA replication. These findings indicate that RITOLS is a physiological mechanism of mtDNA replication, and that it involves a 'bootlace' mechanism, in which processed transcripts are successively hybridized to the lagging-strand template, as the replication fork advances.