Mitochondrial DNA Stability Research Articles

Mfn2R364W, Mfn2G176S, and Mfn2H165R mutations drive Charcot-Marie-Tooth type 2A disease by inducing apoptosis and mitochondrial oxidative phosphorylation damage

Charcot-Marie-Tooth type 2A (CMT2A) is a single-gene motor sensory neuropathy caused by Mfn2 mutation. It is generally believed that CMT2A involves mitochondrial fusion disruption. However, how Mfn2 mutation mediates the mitochondrial membrane fusion loss and its further pathogenic mechanisms remain unclear. Here, in vivo and in vitro mouse models harboring the Mfn2R364W, Mfn2G176S and Mfn2H165R mutations were constructed. Mitochondrial membrane fusion and fission proteins analysis showed that Mfn2R364W, Mfn2G176S, and Mfn2H165R/+ mutations maintain the expression of Mfn2, but promote Drp1 upregulation and Opa1 hydrolytic cleavage. In Mfn2H165R/H165R mutation, Mfn2, Drp1, and Opa1 all play a role in inducing mitochondrial fragmentation, and the mitochondrial aggregation is affected by Mfn2 loss. Further research into the pathogenesis of CMT2A showed these three mutations all induce mitochondria-mediated apoptosis, and mitochondrial oxidative phosphorylation damage. Overall, loss of overall fusion activity affects mitochondrial DNA (mtDNA) stability and causes mitochondrial loss and dysfunction, ultimately leading to CMT2A disease. Interestingly, the differences in the pathogenesis of CMT2A between Mfn2R364W, Mfn2G176S, Mfn2H165R/+ and Mfn2H165R/H165R mutations, including the distribution of Mfn2 and mitochondria, the expression of mitochondrial outer membrane-associated proteins (Bax, VDAC1 and AIF), and the enzyme activity of mitochondrial complex I, are related to the expression of Mfn2.

Three Arabidopsis UMP kinases have different roles in pyrimidine nucleotide biosynthesis and (deoxy)CMP salvage.

Pyrimidine nucleotide monophosphate biosynthesis ends in the cytosol with uridine monophosphate (UMP). UMP phosphorylation to uridine diphosphate (UDP) by UMP KINASEs (UMKs) is required for the generation of all pyrimidine (deoxy)nucleoside triphosphates as building blocks for nucleic acids and central metabolites like UDP-glucose. The Arabidopsis (Arabidopsis thaliana) genome encodes five UMKs and three belong to the AMP KINASE (AMK)-like UMKs, which were characterized to elucidate their contribution to pyrimidine metabolism. Mitochondrial UMK2 and cytosolic UMK3 are evolutionarily conserved, whereas cytosolic UMK1 is specific to the Brassicaceae. In vitro, all UMKs can phosphorylate UMP, cytidine monophosphate (CMP) and deoxycytidine monophosphate (dCMP), but with different efficiencies. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-induced null mutants were generated for UMK1 and UMK2, but not for UMK3, since frameshift alleles were lethal for germline cells. However, a mutant with diminished UMK3 activity showing reduced growth was obtained. Metabolome analyses of germinating seeds and adult plants of single- and higher-order mutants revealed that UMK3 plays an indispensable role in the biosynthesis of all pyrimidine (deoxy)nucleotides and UDP-sugars, while UMK2 is important for dCMP recycling that contributes to mitochondrial DNA stability. UMK1 is primarily involved in CMP recycling. We discuss the specific roles of these UMKs referring also to the regulation of pyrimidine nucleoside triphosphate synthesis.

Single-Nucleotide Polymorphisms in Genes Maintaining the Stability of Mitochondrial DNA Affect the Occurrence, Onset, Severity and Treatment of Major Depressive Disorder.

One of the key features of major depressive disorder (MDD, depression) is increased oxidative stress manifested by elevated levels of mtROS, a hallmark of mitochondrial dysfunction, which can arise from mitochondrial DNA (mtDNA) damage. Thus, the current study explores possibility that the single-nucleotide polymorphisms (SNPs) of genes encoding the three enzymes that are thought to be implicated in the replication, repair or degradation of mtDNA, i.e., POLG, ENDOG and EXOG, have an impact on the occurrence, onset, severity and treatment of MDD. Five SNPs were selected: EXOG c.-188T > G (rs9838614), EXOG c.*627G > A (rs1065800), POLG c.-1370T > A (rs1054875), ENDOG c.-394T > C (rs2977998) and ENDOG c.-220C > T (rs2997922), while genotyping was performed on 538 DNA samples (277 cases and 261 controls) using TaqMan probes. All SNPs of EXOG and ENDOG modulated the risk of depression, but the strongest effect was observed for rs1065800, while rs9838614 and rs2977998 indicate that they might influence the severity of symptoms, and, to a lesser extent, treatment effectiveness. Although the SNP located in POLG did not affect occurrence of the disease, the result suggests that it may influence the onset and treatment outcome. These findings further support the hypothesis that mtDNA damage and impairment in its metabolism play a crucial role not only in the development, but also in the treatment of depression.

Altered H3K4me3 profile at the TFAM promoter causes mitochondrial alterations in preadipocytes from first-degree relatives of type 2 diabetics

BackgroundFirst-degree relatives of type 2 diabetics (FDR) exhibit a high risk of developing type 2 diabetes (T2D) and feature subcutaneous adipocyte hypertrophy, independent of obesity. In FDR, adipose cell abnormalities contribute to early insulin-resistance and are determined by adipocyte precursor cells (APCs) early senescence and impaired recruitment into the adipogenic pathway. Epigenetic mechanisms signal adipocyte differentiation, leading us to hypothesize that abnormal epigenetic modifications cause adipocyte dysfunction and enhance T2D risk. To test this hypothesis, we examined the genome-wide histone profile in APCs from the subcutaneous adipose tissue of healthy FDR.ResultsSequencing-data analysis revealed 2644 regions differentially enriched in lysine 4 tri-methylated H3-histone (H3K4me3) in FDR compared to controls (CTRL) with significant enrichment in mitochondrial-related genes. These included TFAM, which regulates mitochondrial DNA (mtDNA) content and stability. In FDR APCs, a significant reduction in H3K4me3 abundance at the TFAM promoter was accompanied by a reduction in TFAM mRNA and protein levels. FDR APCs also exhibited reduced mtDNA content and mitochondrial-genome transcription. In parallel, FDR APCs exhibited impaired differentiation and TFAM induction during adipogenesis. In CTRL APCs, TFAM-siRNA reduced mtDNA content, mitochondrial transcription and adipocyte differentiation in parallel with upregulation of the CDKN1A and ZMAT3 senescence genes. Furthermore, TFAM-siRNA significantly expanded hydrogen peroxide (H2O2)-induced senescence, while H2O2 did not affect TFAM expression.ConclusionsHistone modifications regulate APCs ability to differentiate in mature cells, at least in part by modulating TFAM expression and affecting mitochondrial function. Reduced H3K4me3 enrichment at the TFAM promoter renders human APCs senescent and dysfunctional, increasing T2D risk.Graphical abstract

Reduced methionine synthase expression results in uracil accumulation in mitochondrial DNA and impaired oxidative capacity.

Adequate thymidylate [deoxythymidine monophosphate (dTMP) or the "T" base in DNA] levels are essential for stability of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Folate and vitamin B12 (B12) are essential cofactors in folate-mediated one-carbon metabolism (FOCM), a metabolic network which supports synthesis of nucleotides (including dTMP) and methionine. Perturbations in FOCM impair dTMP synthesis, causing misincorporation of uracil (or a "U" base) into DNA. During B12 deficiency, cellular folate accumulates as 5-methyltetrahdryfolate (5-methyl-THF), limiting nucleotide synthesis. The purpose of this study was to determine how reduced levels of the B12-dpendent enzyme methionine synthase (MTR) and dietary folate interact to affect mtDNA integrity and mitochondrial function in mouse liver. Folate accumulation, uracil levels, mtDNA content, and oxidative phosphorylation capacity were measured in male Mtr+/+ and Mtr+/- mice weaned onto either a folate-sufficient control (C) diet (2 mg/kg folic acid) or a folate-deficient (FD) diet (lacking folic acid) for 7 weeks. Mtr heterozygosity led to increased liver 5-methyl-THF levels. Mtr+/- mice consuming the C diet also exhibited a 40-fold increase in uracil in liver mtDNA. Mtr+/- mice consuming the FD diet exhibited less uracil accumulation in liver mtDNA as compared to Mtr+/+ mice consuming the FD diet. Furthermore, Mtr+/- mice exhibited 25% lower liver mtDNA content and a 20% lower maximal oxygen consumption rates. Impairments in mitochondrial FOCM are known to lead to increased uracil in mtDNA. This study demonstrates that impaired cytosolic dTMP synthesis, induced by decreased Mtr expression, also leads to increased uracil in mtDNA.

Targeting Mitochondrial Dysfunction in Neurodegenerative Diseases: Expanding the Therapeutic Approaches by Plant-Derived Natural Products.

Mitochondria are the primary source of energy production in neurons, supporting the high energy consumption of the nervous system. Inefficient and dysfunctional mitochondria in the central nervous system have been implicated in neurodegenerative diseases. Therefore, targeting mitochondria offers a new therapeutic opportunity for neurodegenerative diseases. Many recent studies have proposed that plant-derived natural products, as pleiotropic, safe, and readily obtainable sources of new drugs, potentially treat neurodegenerative diseases by targeting mitochondria. In this review, we summarize recent advances in targeting mitochondria in neurotherapeutics by employing plant-derived natural products. We discuss the mechanism of plant-derived natural products according to their mechanism of action on mitochondria in terms of regulating biogenesis, fusion, fission, bioenergetics, oxidative stress, calcium homeostasis, membrane potential, and mitochondrial DNA stability, as well as repairing damaged mitochondria. In addition, we discuss the potential perspectives and challenges in developing plant-derived natural products to target mitochondria, highlighting the clinical value of phytochemicals as feasible candidates for future neurotherapeutics.

EPITHELIAL CELLS OF ULCERATIVE COLITIS PATIENTS REQUIRE NORMAL LEVELS OF MITOCHONDRIA AND BETA-CATENIN TO UTILIZE BUTYRATE METABOLISM AND PROMOTE ULCER HEALING

Abstract BACKGROUND Under normal conditions butyrate produced by obligate anaerobes (e.g. firmicutes) provides the dominant energy source for colonocytes via β-oxidation and the tricarboxylic acid cycle. Ulcerative Colitis (UC) is an inflammatory bowel disease (IBD) characterized by reduced butyrate metabolism and intestinal epithelial cell (IEC) mitochondria (needed for β-oxidation). Given that nonhealing ulcers in UC are associated with reduced crypt fissioning and branching, we asked whether there was a relationship between mitochondrial deficiency and butyrate-induced crypt branching using colonoid cultures grown from human biopsies. METHODS IECs from biopsies taken from active and uninvolved areas of the same UC patient were grown in colonoid cultures. After the second passage, the colonoids with crypt branching were quantified. In separate studies, we modeled mitochondrial deficiency by shRNA knockdown of transcription mitochondrial factor A (Tfam) in human colonoids, a gene responsible for mitochondrial DNA stability and transcription. Both shControl and shTfam colonoids were treated with 5mM of Na-butyrate overnight. NCM460 cells were also treated with 5mM of butyrate overnight, and both ATP levels and TCF/LEF (b-catenin transcriptional activity) were examined. RESULTS Colonoids from active UC segments demonstrated 27% less crypt branching than uninvolved areas. These areas also displayed reduced mitochondria complex levels by WB of IEC. To directly examine the role of mitochondria in these effects, crypt branching was examined in shTFAM-treated colonoids where crypt branching levels were 31% less than shCont-treated colonoids (Fig. 1). Butyrate treatment of both shCont and shTfam colonoids increased crypt formation by 11% and 8%, respectively compared to untreated groups. In addition, butyrate increased levels of ATP by 55% and b-catenin transcriptional activity (TCF-LEF) by >600%. CONCLUSION This data was consistent with the notion that the failure to heal ulceration in UC patients involves both 1) reduction in butyrate availability from the microbiome (ie reduced stimulation) and 2) reduced mitochondria needed to metabolize butyrate. Upon effective anti-inflammatory treatment, we believe butyrate levels and IEC mitochondrial metabolism increase which contributes to IEC b-catenin signaling and enhanced crypt fissioning/branching needed for effective ulcer healing. We believe this data provide a potential mechanism for how enhanced mitochondrial function contributes to ulcer healing in IBD.

Mitochondrial genome undergoes de novo DNA methylation that protects mtDNA against oxidative damage during the peri-implantation window.

Mitochondrial remodeling during the peri-implantation stage is the hallmark event essential for normal embryogenesis. Among the changes, enhanced oxidative phosphorylation is critical for supporting high energy demands of postimplantation embryos, but increases mitochondrial oxidative stress, which in turn threatens mitochondrial DNA (mtDNA) stability. However, how mitochondria protect their own histone-lacking mtDNA, during this stage remains unclear. Concurrently, the mitochondrial genome gain DNA methylation by this stage. Its spatiotemporal coincidence with enhanced mitochondrial stress led us to ask if mtDNA methylation has a role in maintaining mitochondrial genome stability. Herein, we report that mitochondrial genome undergoes de novo mtDNA methylation that can protect mtDNA against enhanced oxidative damage during the peri-implantation window. Mitochondrial genome gains extensive mtDNA methylation during transition from blastocysts to postimplantation embryos, thus establishing relatively hypermethylated mtDNA from hypomethylated state in blastocysts. Mechanistic study revealed that DNA methyltransferase 3A (DNMT3A) and DNMT3B enter mitochondria during this process and bind to mtDNA, via their unique mitochondrial targeting sequences. Importantly, loss- and gain-of-function analyses indicated that DNMT3A and DNMT3B are responsible for catalyzing de novo mtDNA methylation, in a synergistic manner. Finally, we proved, invivo and invitro, that increased mtDNA methylation functions to protect mitochondrial genome against mtDNA damage induced by increased mitochondrial oxidative stress. Together, we reveal mtDNA methylation dynamics and its underlying mechanism during the critical developmental window. We also provide the functional link between mitochondrial epigenetic remodeling and metabolic changes, which reveals a role for nuclear-mitochondrial crosstalk in establishing mitoepigenetics and maintaining mitochondrial homeostasis.

Molecular signature of cardiac remodeling associated with Polymerase Gamma mutation

AimsMetabolic function/dysfunction is central to aging biology. This is well illustrated by the Polymerase Gamma (POLG) mutant mouse where a key residue of the mitochondrial DNA polymerase is mutated (D257A), causing loss of mitochondrial DNA stability and dramatically accelerated aging processes. Given known cardiac phenotypes in the POLG mutant, we sought to characterize the course of cardiac dysfunction in the POLG mutant to guide future intervention studies. Materials and methodsCardiac echocardiography and terminal hemodynamic analyses were used to define the course of dysfunction in the right and left cardiac ventricles in the POLG mutant. We also conducted RNA-seq analysis on cardiac right ventricles to identify mechanisms engaged by severe metabolic dysfunction and compared this analysis to several publically available datasets. Key findingsInteresting sex differences were noted as female POLG mutants died earlier than male POLG mutants and LV chamber diameters were impacted earlier in females than males. Moreover, male mutants showed LV wall thinning while female mutant LV walls were thicker. Both males and females displayed significant RV hypertrophy. POLG mutants displayed a gene expression pattern associated with inflammation, fibrosis, and heart failure. Finally, comparative omics analyses of publically available data provide additional mechanistic and therapeutic insights. SignificanceAging-associated cardiac dysfunction is a growing clinical problem. This work uncovers sex-specific cardiac responses to severe metabolic dysfunction that are reminiscent of patterns seen in human heart failure and provides insights to the molecular mechanisms engaged downstream of severe metabolic dysfunction that warrant further investigation.

An HSP90 cochaperone Ids2 maintains the stability of mitochondrial DNA and ATP synthase

BackgroundProteostasis unbalance and mitochondrial dysfunction are two hallmarks of aging. While the chaperone folds and activates its clients, it is the cochaperone that determines the specificity of the clients. Ids2 is an HSP90’s cochaperone controlling mitochondrial functions, but no in vivo clients of Ids2 have been reported yet.ResultsWe performed a screen of the databases of HSP90 physical interactors, mitochondrial components, and mutants with respiratory defect, and identified Atp3, a subunit of the complex V ATP synthase, as a client of Ids2. Deletion of IDS2 destabilizes Atp3, and an α-helix at the middle region of Ids2 recruits Atp3 to the folding system. Shortage of Ids2 or Atp3 leads to the loss of mitochondrial DNA. The intermembrane space protease Yme1 is critical to maintaining the Atp3 protein level. Moreover, Ids2 is highly induced when cells carry out oxidative respiration.ConclusionsThese findings discover a cochaperone essentially for maintaining the stability of mitochondrial DNA and the proteostasis of the electron transport chain—crosstalk between two hallmarks of aging.

Assessment of Mitochondrial DNA Copy Number, Stability, and Repair in Arabidopsis.

Mitochondrial functions depend on the proper maintenance and expression of the mitochondrial genome (mtDNA). Therefore, understanding mtDNA replication and repair requires methods to assess its integrity. Mutations or chemical treatments that affect processes involved in the maintenance or stability of the mtDNA can affect its global copy number, but also the relative abundance of different genomic regions or the frequency of illegitimate recombination across repeated sequences. These can be conveniently tested by quantitative PCR (qPCR). Arabidopsis thaliana offers several advantages for studying these processes, because of the extensive collections of mutants, natural accessions and other genetic resources available from stock centers. Here we describe protocols we routinely use to explore changes in mtDNA copy number and relative stoichiometry in Arabidopsis mutants of genes involved in the replication, repair and recombination of the mtDNA.

An HSP90 Cochaperone Ids2 Maintains the Stability of Mitochondrial DNA and ATP Synthase

A previous study demonstrates crosstalk among three hallmarks of aging: deregulated nutrient sensing, loss of proteostasis, and mitochondrial dysfunction. Nutrient overconsumption impairs the folding activity of the HSP90-Ids2 complex, but it remains unclear through what mechanisms proteostasis controls mitochondrial functions. Here we uncover Ids2 as an essential cochaperone for maintaining mitochondrial DNA and functions. A screen of the databases of Hsc82 physical interactors, mitochondrial components, and mutants with respiratory defect identified Atp3, a subunit of the complex V ATP synthase, as a client of Ids2. Deletion of IDS2 destabilizes Atp3, and an a-helix at the middle region of Ids2 recruits Atp3 to the folding system. Shortage of Ids2 or Atp3 leads to the loss of mitochondrial DNA. The intermembrane space protease Yme1 is critical for Atp3's quality control. Moreover, Ids2 is highly induced when cells carry out oxidative respiration. In summary, we identify the first folding client of Ids2 and explain why the deficiency of Ids2 triggers mitochondrial DNA loss and mitochondrial dysfunction.

The Vacuole and Mitochondria Patch (vCLAMP) Protein Mcp1 Is Involved in Maintenance of Mitochondrial Function and Mitophagy in Candida albicans.

The vacuole and mitochondria patches (vCLAMPs) are novel membrane contact sites in yeast. However, their role in autophagy has not been elucidated so far. In this article, the role of Mcp1, one core component of vCLAMP, in mitophagy of Candida albicans was investigated. Deletion of MCP1 led to abnormal accumulation of enlarged mitochondria and attenuated stability of mitochondrial DNA (mtDNA) in C. albicans when cultured in non-fermentable carbon sources. Furthermore, the mcp1Δ/Δ mutant exhibited defective growth and degradation of Csp37-GFP. These results indicate that Mcp1 plays a crucial role in mitophagy and maintenance of mitochondrial functions under the non-fermentable condition. Interestingly, this deletion had no impact on degradation of Atg8 (the macroautophagy reporter) and Lap41 (the cytoplasm-to-vacuole targeting pathway marker) under SD-N medium. Moreover, deletion of MCP1 inhibited filamentous growth and impaired virulence of the pathogen. This study provides an insight to vCLAMPs in cellular functions and pathogenicity in C. albicans.

Effectiveness of various methods of DNA isolation from bones and teeth of animals exposed to high temperature

In the event of fires, natural disasters, and other events associated with high temperature, bones and teeth are the only source of genetic material for identifying human or animal carcasses. To obtain reliable final results of identification tests, the use of appropriate nucleic acid extraction methods is crucial. Therefore, the main objective of this research was to evaluate the effectiveness of selected methods of DNA isolation from animal burnt bones and teeth. In addition, the effect of the duration of high temperature on the stability of nuclear and mitochondrial DNA in these tissues was determined, as well as the possibility of using the genetic material obtained for species identification of remains of unknown origin. Bones and teeth collected during necropsy of dogs were burnt in a laboratory oven at 400 °C (752 °F; 673.15 K) for 5, 10, 15, 30, 45 and 60 min. DNA was isolated according to four different protocols, using three commercial kits, i.e. the PrepFiler® Forensic DNA Extraction Kit from Applied Biosystems, the QIAamp® DNA Investigator Kit from QIAGEN, and the DNA Mini Kit from Syngen, as well as a classic organic method. The effectiveness of these methods was compared by assessing the amount of isolated DNA using Real-Time PCR and its purity using a NanoDrop™ spectrophotometer. Each isolate was also subjected to PCR with primers designed to amplify fragments of dog mitochondrial DNA. The effectiveness of species identification was assessed for the method showing the best DNA recovery and for the organic method, considered the gold standard for analysis of difficult material. The QIAamp® DNA Investigator Kit showed the highest efficiency of DNA isolation from bones and teeth burnt for 15 min (the longest burning time for which DNA could still be recovered from bones and teeth). The results of the experiment clearly indicate that DNA stability in hard tissues depends on how long they burn. In the case of exposure to 400 °C, reliable genetic testing, including species identification, is possible when the burning time does not exceed 15 min. Among the hard tissues examined, bones proved more suitable than teeth for identification purposes. It was also concluded that identification of bone remains with extreme heat damage should be based on mitochondrial DNA analysis.

Functional analysis of a novel POLγA mutation associated with a severe perinatal mitochondrial encephalomyopathy

Mutations in the mitochondrial DNA polymerase gamma catalytic subunit (POLγA) compromise the stability of mitochondrial DNA (mtDNA) by leading to mutations, deletions and depletions in mtDNA. Patients with mutations in POLγA often differ remarkably in disease severity and age of onset. In this work we have studied the functional consequence of POLγA mutations in a patient with an uncommon and a very severe disease phenotype characterized by prenatal onset with intrauterine growth restriction, lactic acidosis from birth, encephalopathy, hepatopathy, myopathy, and early death. Muscle biopsy identified scattered COX-deficient muscle fibers, respiratory chain dysfunction and mtDNA depletion. We identified a novel POLγA mutation (p.His1134Tyr) in trans with the previously identified p.Thr251Ile/Pro587Leu double mutant. Biochemical characterization of the purified recombinant POLγA variants showed that the p.His1134Tyr mutation caused severe polymerase dysfunction. The p.Thr251Ile/Pro587Leu mutation caused reduced polymerase function in conditions of low dNTP concentration that mimic postmitotic tissues. Critically, when p.His1134Tyr and p.Thr251Ile/Pro587Leu were combined under these conditions, mtDNA replication was severely diminished and featured prominent stalling. Our data provide a molecular explanation for the patient´s mtDNA depletion and clinical features, particularly in tissues such as brain and muscle that have low dNTP concentration.

Mitochondrial Oxidative Stress Induces Rapid Intermembrane Space/Matrix Translocation of Apurinic/Apyrimidinic Endonuclease 1 Protein through TIM23 Complex

Mitochondria are essential cellular organelles that import the majority of proteins to sustain their function in cellular metabolism and homeostasis. Due to their role in oxidative phosphorylation, mitochondria are constantly affected by oxidative stress. Stability of mitochondrial DNA (mtDNA) is essential for mitochondrial physiology and cellular well-being and for this reason mtDNA lesions have to be rapidly recognized and repaired. Base excision repair (BER) is the main pathway responsible for repairing non-helix distorting base lesions both into the nucleus and in mitochondria. Apurinic/Apyrimidinic Endonuclease 1 (APE1) is a key component of BER pathway and the only protein that can recognize and process an abasic (AP) site. Comprehensions of the mechanisms regulating APE1 intracellular trafficking are still fragmentary. In this study we focused our attention on the mitochondrial form of APE1 protein and how oxidative stress induces its translocation to maintain mtDNA integrity. Our data proved that: (i) the rise of mitochondrial ROS determines a very rapid translocation of APE1 from the intermembrane space (IMS) into the matrix; and (ii) TIM23/PAM machinery complex is responsible for the matrix translocation of APE1. Moreover, our data support the hypothesis that the IMS, where the majority of APE1 resides, could represent a sort of storage site for the protein.

Irc3 is a monomeric DNA branch point-binding helicase in mitochondria of the yeast Saccharomycescerevisiae.

Irc3 is a superfamily II DNA helicase required for the maintenance of mitochondrial DNA stability in Saccharomycescerevisiae. Here, we show that recombinant Irc3 is a monomeric protein and that it can form a binary complex with forked DNA. The catalytically active enzyme is a monomer as no positive cooperativity of ATP hydrolysis or DNA unwinding can be detected. Interestingly, we find that Irc3 prefers to unwind the nascent lagging strand at a replication fork. Using DNase I footprinting, we demonstrate that Irc3 captures DNA substrates by establishing a strong contact at the DNA branching point. Additional protections on the lagging strand template suggest a 3'-to-5' polarity for Irc3 movement.

Epigenetics and Mitochondrial Stability in the Metabolic Memory Phenomenon Associated with Continued Progression of Diabetic Retinopathy

Retinopathy continues to progress even when diabetic patients try to control their blood sugar, but the molecular mechanism of this ‘metabolic memory’ phenomenon remains elusive. Retinal mitochondria remain damaged and vicious cycle of free radicals continues to self-propagate. DNA methylation suppresses gene expression, and diabetes activates DNA methylation machinery. Our aim was to investigate the role of DNA methylation in continued compromised mitochondrial dynamics and genomic stability in diabetic retinopathy. Using retinal endothelial cells, incubated in 20 mM glucose for four days, followed by 5 mM glucose for four days, and retinal microvessels from streptozotocin-induced diabetic rats in poor glycemia for four months, followed by normal glycemia for four additional months, DNA methylation of mitochondrial fusion and mismatch repair proteins, Mfn2 and Mlh1 respectively, was determined. Retinopathy was detected in trypsin-digested microvasculature. Re-institution of good glycemia had no beneficial effect on hypermethylation of Mfn2 and Mlh1 and retinal function (electroretinogram), and the retinopathy continued to progress. However, intervention of good glycemia directly with DNA methylation inhibitors (Azacytidine or Dnmt1-siRNA), prevented Mfn2 and Mlh1 hypermethylation, and ameliorated retinal dysfunction and diabetic retinopathy. Thus, direct regulation of DNA methylation can prevent/reverse diabetic retinopathy by maintaining mitochondrial dynamics and DNA stability, and prevent retinal functional damage.

A stable tetramer is not the only oligomeric state that mitochondrial single-stranded DNA binding proteins can adopt

Mitochondrial single-stranded DNA (ssDNA)-binding proteins (mtSSBs) are required for mitochondrial DNA replication and stability and are generally assumed to form homotetramers, and this species is proposed to be the one active for ssDNA binding. However, we recently reported that the mtSSB from Saccharomyces cerevisiae (ScRim1) forms homotetramers at high protein concentrations, whereas at low protein concentrations, it dissociates into dimers that bind ssDNA with high affinity. In this work, using a combination of analytical ultracentrifugation techniques and DNA binding experiments with fluorescently labeled DNA oligonucleotides, we tested whether the ability of ScRim1 to form dimers is unique among mtSSBs. Although human mtSSBs and those from Schizosaccharomyces pombe, Xenopus laevis, and Xenopus tropicalis formed stable homotetramers, the mtSSBs from Candida albicans and Candida parapsilosis formed stable homodimers. Moreover, the mtSSBs from Candida nivariensis and Candida castellii formed tetramers at high protein concentrations, whereas at low protein concentrations, they formed dimers, as did ScRim1. Mutational studies revealed that the ability to form either stable tetramers or dimers depended on a complex interplay of more than one amino acid at the dimer-dimer interface and the C-terminal unstructured tail. In conclusion, our findings indicate that mtSSBs can adopt different oligomeric states, ranging from stable tetramers to stable dimers, and suggest that a dimer of mtSSB may be a physiologically relevant species that binds to ssDNA in some yeast species.

Enzymology of mitochondrial DNA repair.

The mitochondrial genome encodes proteins essential for the oxidative phosphorylation and, consequently, for proper mitochondrial function. Its localization and, possibly, structural organization contribute to higher DNA damage accumulation, when compared to the nuclear genome. In addition, the mitochondrial genome mutates at rates several times higher than the nuclear, although the causal relationship between these events are not clearly established. Maintaining mitochondrial DNA stability is critical for cellular function and organismal fitness, and several pathways contribute to that, including damage tolerance and bypass, degradation of damaged genomes and DNA repair. Despite initial evidence suggesting that mitochondria lack DNA repair activities, most DNA repair pathways have been at least partially characterized in mitochondria from several model organisms, including humans. In this chapter, we review what is currently known about how the main DNA repair pathways operate in mitochondria and contribute to mitochondrial DNA stability, with focus on the enzymology of mitochondrial DNA repair.