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.

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.

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.

Clinical efficacy of dissolvable microneedles armed with anti-melanogenic compounds to counter hyperpigmentation.

BACKGROUND: Hyperpigmentation is a complex physiological process associated with alterations of skin color due to melanin overproduction and distribution. Both intrinsic and extrinsic factors influence discoloration with the involvement of multiple pathways in cooperative manners. Restoring natural skin color requires a multi-targeted approach. Latest advances in anti-melanogenic compounds and microneedle delivery system have presented opportunities for tackling hyperpigmentationsuccessfully. AIMS: This work was aimed at assessing the dermal tolerability and efficacy of hyaluronic acid-based microneedle patches loaded with anti-melanogenic actives for improvements of skin discoloration. PATIENTS/METHODS: The test products consist of hyaluronic acid with niacinamide, ascorbic acid 2-glucoside, tranexamic acid, resveratrol, 4-n-butyl-resorcinol, and Halidrys siliquosa extract. In a monocentric 12 weeks clinical trial, the HA-MNs patches were applied to the affected areas of the faces on subjects with hyperpigmented skin. The color properties of the skin were analyzed spectrophotometrically. RESULTS: The products were tolerated remarkably; none of the subjects informed any primary or cumulative skin responses. Evaluation of color related measurable skin properties provided insight into the general effectiveness: The individual topology measurements showed 51.4% noticeable improvements in hyperpigmented zones, which were also in good agreement with the personal impressions of the subjects determined by questionnaires before and after the treatments. CONCLUSIONS: The concentrated blends of actives in microneedle patches act in a multi-targeted manner, and they could be worked in a complementary fashion for the improvement of skin color and appearance. The study has established the overall convenience of the HA-MNs with the formulation of anti-melogenic compounds against skin discoloration.

Efficacy of bioactive peptides loaded on hyaluronic acid microneedle patches: A monocentric clinical study.

BACKGROUND: Aging skin is a gradual physiological process associated with functional and structural changes of the skin. Both intrinsic and extrinsic factors influence skin aging by the involvement of multiple pathways. Restoring natural skin conditions requires a multi-targeted approach. Recent developments in both bioactive peptides and microneedle delivery system have presented an opportunity for tackling premature skin aging. AIMS: This study was aimed at evaluating the dermal tolerability and efficacy of hyaluronic acid-based microneedle patches loaded with bioactives for restoration of the skin properties including hydration, wrinkle reduction, density, and thickness. PATIENTS/METHODS: The test product of HA-MNs comprises arginine/lysine polypeptide, acetyl octapeptide-3, palmitoyl tripeptide-5, adenosine, and seaweed extracts. In the monocentric 12-week clinical trial, the HA-MNs patches were applied to the outer corner of the right and left eye and a defined area on the volar forearm on healthy subjects with aged skin. Instrumental analysis of skin properties was determined. RESULTS: The product was tolerated excellently; none of the subjects reported any primary or cumulative skin reactions. Assessment of measurable skin properties provides insight into the general effectiveness: The fine lines/wrinkles showed 25.8% noticeable decrease; the skin hydration measurements demonstrated 15.4% improvement; the skin density and thickness in the dermis increased 14.2% and 12.9%, respectively. CONCLUSIONS: The composition of the microneedle patches works in a multi-targeted manner and all ingredients might possibly be acted synergistically for the improvement of skin structure, function, and appearance. The study has demonstrated the overall usefulness of the HA-MNs with careful formulation for skin care applications.

MCM-GINS and MCM-MCM interactions in vivo visualised by bimolecular fluorescence complementation in fission yeast.

BACKGROUND: Each of the three individual components of the CMG complex (Cdc45, MCM and GINS) is essential for chromosomal DNA replication in eukaryotic cells, both for the initiation of replication at origins and also for normal replication fork progression. The MCM complex is a DNA helicase that most likely functions as the catalytic core of the replicative helicase, unwinding the parental duplex DNA ahead of the moving replication fork, whereas Cdc45 and the GINS complex are believed to act as accessory factors for MCM. RESULTS: To investigate interactions between components of the CMG complex, we have used bimolecular fluorescence complementation (BiFC) in the fission yeast Schizosaccharomyces pombe for the first time, to analyse protein-protein interactions between GINS and MCM subunits expressed from their native chromosomal loci. We demonstrate interactions between GINS and MCM in the nuclei of exponentially-growing fission yeast cells and on chromatin in binucleate S-phase cells. In addition we present evidence of MCM-MCM interactions in diploid fission yeast cells. As with GINS-MCM interactions, MCM-MCM interactions also occur on chromatin in S-phase cells. CONCLUSION: Bimolecular fluorescence complementation can be used in fission yeast to visualise interactions between two of the three components of the CMG complex, offering the prospect that this technique could in the future be used to allow studies on replication protein dynamics in living S. pombe cells.

Nickel resistance in fission yeast associated with the magnesium transport system.

We isolated and characterized a nickel (Ni2+)-resistant mutant (GA1) of Schizosaccharomyces pombe. This mutant strain displayed resistance to both Ni2+ and Zn2+, but not to Cd2+, Co2+, and Cu2+. The growth rate of GA1 increased proportionally with increasing Mg2+ concentrations until 50 mM Mg2+. The GA1 mutation phenotype suggests a defect in Mg2+ uptake. Sequence analysis of the GA1 open reading frame (ORF) O13779, which is homologous to the prokaryotic and eukaryotic CorA Mg2+ transport systems, revealed a point mutation at codon 153 (ccc to acc) resulting in a Pro153Thr substitution in the N-terminus of the CorA domain. Our results provide novel genetic information about Ni2+ resistance in fission yeast. Specifically, that reducing Mg2+ influx through the CorA Mg2+ transport membrane protein confers Ni2+ resistance in S. pombe. We also report that Ni2+ ion detoxification of the fission yeast is related to histidine metabolism and pH.