Structural modifications induced by the mtDBP-C protein in the replication origin of Xenopus laevis mitochondrial DNA

The structure of the non-coding region of Xenopus laevis mitochondrial DNA has been studied by electron microscopy analysis of DNA molecules end-labelled with streptavidin-ferritin. We have shown that the effect of a protein modifying the shape of the DNA double-helix can be studied and precisely located by this method. It was found that the non-coding region contains curved segments and that the mitochondrial protein mtDBP-C preferentially enhances the curvature of the promoters-replication origin region.     

Mitochondrial DNA ligase function in Saccharomyces cerevisiae

The Saccharomyces cerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the nucleus and the mitochondria. While nuclear Cdc9p is known to play an essential role in nuclear DNA replication and repair, its role in mitochondrial DNA dynamics has not been defined. It is also unclear whether additional DNA ligase proteins are present in yeast mitochondria. To address these issues, mitochondrial DNA ligase function in S.cerevisiae was analyzed. Biochemical analysis of mitochondrial protein extracts supported the conclusion that Cdc9p was the sole DNA ligase protein present in this organelle. Inactivation of mitochondrial Cdc9p function led to a rapid decline in cellular mitochondrial DNA content in both dividing and stationary yeast cultures. In contrast, there was no apparent defect in mitochondrial DNA dynamics in a yeast strain deficient in Dnl4p (Deltadnl4). The Escherichia coli ECO:RI endonuclease was targeted to yeast mitochondria. Transient expression of this recombinant ECO:RI endonuclease led to the formation of mitochondrial DNA double-strand breaks. While wild-type and Deltadnl4 yeast were able to rapidly recover from this mitochondrial DNA damage, clones deficient in mitochondrial Cdc9p were not. These results support the conclusion that yeast rely upon a single DNA ligase, Cdc9p, to carry out mitochondrial DNA replication and recovery from both spontaneous and induced mitochondrial DNA damage.     

Amino terminal sequence of the mitochondrial protein mtDBP-C: similarity with nonhistone chromosomal proteins HMG 1 and 2.

We have previously reported the characterization of a DNA-binding protein isolated from Xenopus laevis mitochondria (mtDBP-C). The amino terminal sequence of this protein (26 residues) has been determined by automated Edman degradation and used to search for sequence similarity with the NBRF library. A segment of 17 amino acids displays 47.1% of identity with proteins HMG-1 and 2 of various vertebrate species.     

Mitochondrial translocation of APE1 relies on the MIA pathway

APE1 is a multifunctional protein with a fundamental role in repairing nuclear and mitochondrial DNA lesions caused by oxidative and alkylating agents. Unfortunately, comprehensions of the mechanisms regulating APE1 intracellular trafficking are still fragmentary and contrasting. Recent data demonstrate that APE1 interacts with the mitochondrial import and assembly protein Mia40 suggesting the involvement of a redox-assisted mechanism, dependent on the disulfide transfer system, to be responsible of APE1 trafficking into the mitochondria. The MIA pathway is an import machinery that uses a redox system for cysteine enriched proteins to drive them in this compartment. It is composed by two main proteins: Mia40 is the oxidoreductase that catalyzes the formation of the disulfide bonds in the substrate, while ALR reoxidizes Mia40 after the import. In this study, we demonstrated that: (i) APE1 and Mia40 interact through disulfide bond formation; and (ii) Mia40 expression levels directly affect APE1''s mitochondrial translocation and, consequently, play a role in the maintenance of mitochondrial DNA integrity. In summary, our data strongly support the hypothesis of a redox-assisted mechanism, dependent on Mia40, in controlling APE1 translocation into the mitochondrial inner membrane space and thus highlight the role of this protein transport pathway in the maintenance of mitochondrial DNA stability and cell survival.     

DNA biosynthesis in rat liver mitochondria: Inhibition by sulfhydryl compounds and stimulation by cytoplasmic proteins

The sulfhydryl compounds, 2-mercaptoethanol, dithiothreitol, cysteine. and glutathione inhibit the incorporation of [³H]dTTP or [³H]dATP into mitochondrial DNA by rat liver mitochondria in vitro. The lack of inhibition by non-SH-containing analogs indicates that the SH group is responsible for the inhibition.The inhibition does not result from an effect of the sulfhydryl compounds on precursor permeability, ATP formation, or respiration, or the action of the thiol on the outer mitochondrial membrane. An intact inner membrane is not required for the action of the inhibitor. Furthermore, SH compounds do not appear to exert their effect by activation of a mitochondrial nuclease, chemical breakdown of high molecular-weight mitochondrial DNA or dissociation of membrane-bound DNA from the inner mitochondrial membrane. Incorporation of labeled precursor into DNA by mitochondrial DNA polymerase, when removed from the inner mitochondrial membrane, is not inhibited by SH compounds.Cytoplasmic extracts prepared from rat and mouse tumors and 22-h regenerating rat liver contain a protein(s) not detectable in normal rat liver which can reverse the inhibition by SH compounds of the synthesis of mitochondrial DNA in rat liver mitochondria in vitro.More importantly, when the stimulatory protein(s) is partially purified by affinity chromatography on DNA-cellulose, it is possible to demonstrate that this protein(s) also stimulates the synthesis of mitochondrial DNA by normal rat liver mitochondria in vitro in the absence of the sulfhydryl inhibitor.     

The mitochondrial DNA helicase TWINKLE can assemble on a closed circular template and support initiation of DNA synthesis

Mitochondrial DNA replication is performed by a simple machinery, containing the TWINKLE DNA helicase, a single-stranded DNA-binding protein, and the mitochondrial DNA polymerase γ. In addition, mitochondrial RNA polymerase is required for primer formation at the origins of DNA replication. TWINKLE adopts a hexameric ring-shaped structure that must load on the closed circular mtDNA genome. In other systems, a specialized helicase loader often facilitates helicase loading. We here demonstrate that TWINKLE can function without a specialized loader. We also show that the mitochondrial replication machinery can assemble on a closed circular DNA template and efficiently elongate a DNA primer in a manner that closely resembles initiation of mtDNA synthesis in vivo.     

Mitochondrial RNase H1 activity regulates R-loop homeostasis to maintain genome integrity and enable early embryogenesis in Arabidopsis

Plant mitochondrial genomes undergo frequent homologous recombination (HR). Ectopic HR activity is inhibited by the HR surveillance pathway, but the underlying regulatory mechanism is unclear. Here, we show that the mitochondrial RNase H1 AtRNH1B impairs the formation of RNA:DNA hybrids (R-loops) and participates in the HR surveillance pathway in Arabidopsis thaliana. AtRNH1B suppresses ectopic HR at intermediate-sized repeats (IRs) and thus maintains mitochondrial DNA (mtDNA) replication. The RNase H1 AtRNH1C is restricted to the chloroplast; however, when cells lack AtRNH1B, transport of chloroplast AtRNH1C into the mitochondria secures HR surveillance, thus ensuring the integrity of the mitochondrial genome and allowing embryogenesis to proceed. HR surveillance is further regulated by the single-stranded DNA-binding protein ORGANELLAR SINGLE-STRANDED DNA BINDING PROTEIN1 (OSB1), which decreases the formation of R-loops. This study uncovers a facultative dual targeting mechanism between organelles and sheds light on the roles of RNase H1 in organellar genome maintenance and embryogenesis.

This study clarifies the function of mitochondrial RNase H1 in genome stability and early embryogenesis in plants, and shows that mitochondrial R-loops are involved in homologous recombination surveillance of mtDNA. Facultative re-targeting of the chloroplast RNase H1 protein to mitochondria, in response to cellular conditions, can help guarantee mitochondrial RNase H1 activity.     

Nitric oxide protects against mitochondrial permeabilization induced by glutathione depletion: role of S-nitrosylation?

Nitric oxide (NO) is known to mediate a multitude of biological effects including inhibition of respiration at cytochrome c oxidase (COX), formation of peroxynitrite (ONOO-) by reaction with mitochondrial superoxide (O2*-), and S-nitrosylation of proteins. In this study, we investigated pathways of NO metabolism in lymphoblastic leukemic CEM cells in response to glutathione (GSH) depletion. We found that NO blocked mitochondrial protein thiol oxidation, membrane permeabilization, and cell death. The effects of NO were: (1) independent of respiratory chain inhibition since protection was also observed in CEM cells lacking mitochondrial DNA (rho0) which do not possess a functional respiratory chain and (2) independent of ONOO- formation since nitrotyrosine (a marker for ONOO- formation) was not detected in extracts from cells treated with NO after GSH depletion. However, NO increased the level of mitochondrial protein S-nitrosylation (SNO) determined by the Biotin Switch assay and by the release of NO from mitochondrial fractions treated with mercuric chloride (which cleaves SNO bonds to release NO). In conclusion, these results indicate that NO blocks cell death after GSH depletion by preserving the redox status of mitochondrial protein thiols probably by a mechanism that involves S-nitrosylation of mitochondrial protein thiols.     

Studies of mitochondrial development prior to lactogenesis in the mouse mammary gland

Studies were performed to define mitochondrial development in relation to epithelial cell proliferation and differentiation prior to lactogenesis in the mammary gland of the mouse. Gland weight, total DNA, and total protein, selected as criteria of gland growth and proliferation, and various parameters of mitochondrial development were followed throughout the period. Gland weight and total DNA started increasing about Day 5 of pregnancy and reached maximal values by parturition. Total gland protein began to increase at the same time but did not reach maximal values until Day 4 of lactation. Total mitochondrial protein and the mitochondrial marker enzyme activities, succinate oxidase, succinate-linked ATP formation, and cytochrome oxidase increased gradually during pregnancy with rapid 2- to 3-fold increases occurring during the early days of lactation. Similarly, succinate oxidase activity per unit DNA of isolated mammary parenchymal cells increased gradually from mid-pregnancy to parturition with a precipitous, 2-fold increase occurring during early lactation.     

Mitochondrial DNA ligase III function is independent of Xrcc1

Hamster EM9 cells, which lack Xrcc1 protein, have reduced levels of DNA ligase III and are defective in nuclear base excision repair. The Xrcc1 protein stabilizes DNA ligase III and may even play a direct role in catalyzing base excision repair. Since DNA ligase III is also thought to function in mitochondrial base excision repair, it seemed likely that mitochondrial DNA ligase III function would also be dependent upon Xrcc1. However, several lines of evidence indicate that this is not the case. First, western blot analysis failed to detect Xrcc1 protein in mitochondrial extracts. Second, DNA ligase III levels present in mitochondrial protein extracts from EM9 cells were indistinguishable from those seen in similar extracts from wild-type (AA8) cells. Third, the mitochondrial DNA content of both cell lines was identical. Fourth, EM9 cells displayed no defect in their ability to repair spontaneous mitochondrial DNA damage. Fifth, while EM9 cells were far more sensitive to the cytotoxic effects of ionizing radiation due to a defect in nuclear DNA repair, there was no apparent difference in the ability of EM9 and AA8 cells to restore their mitochondrial DNA to pre-irradiation levels. Thus, mitochondrial DNA ligase III function is independent of the Xrcc1 protein.     

DNA-binding proteins of mammalian mitochondria

Acid-soluble proteins were isolated from liver and spleen mitochondria and their ability to form complexes with DNA was investigated. According to electrophoresis data, acid-soluble proteins include about 20 polypeptides ranging in the molecular mass from 10 to 120 kDa. It was found that acid-soluble proteins form stable DNA-protein complexes at a physiological NaCl concentration. Different polypeptides possess different degrees of DNA affinity. There is no significant difference between DNA-binding proteins of mitochondria from liver and those from spleen as to their ability to form complexes with mtDNA and nDNA. In the presence of 5 microg of DNA most polypeptides were bound to DNA, and further increase in DNA amount affected little the binding of proteins to DNA. There was no distinct difference in DNA-protein complex formation of liver mitochondrial acid-soluble proteins with nDNA or mtDNA. Also, it was detected that with these mitochondrial acid-soluble proteins, proteases that specifically cleave these proteins are associated. It was shown for the first time that these proteases are activated by DNA. DNA-binding proteins including DNA-activated mitochondrial proteases are likely to participate in the regulation of the structural organization and functional activity of mitochondrial DNA.     

The human DNA ligase III gene encodes nuclear and mitochondrial proteins

We provide evidence that the human DNA ligase III gene encodes a mitochondrial form of this enzyme. First, the DNA ligase III cDNA contains an in-frame ATG located upstream from the putative translation initiation start site. The DNA sequence between these two ATG sites encodes an amphipathic helix similar to previously identified mitochondrial targeting peptides. Second, recombinant green fluorescent protein harboring this sequence at its amino terminus was efficiently targeted to the mitochondria of Cos-1 monkey kidney cells. In contrast, native green fluorescent protein distributed to the cytosol. Third, a series of hemagglutinin-DNA ligase III minigene constructs were introduced into Cos-1 cells, and immunocytochemistry was used to determine subcellular localization of the epitope-tagged DNA ligase III protein. These experiments revealed that inactivation of the upstream ATG resulted in nuclear accumulation of the DNA ligase III protein, whereas inactivation of the downstream ATG abolished nuclear localization and led to accumulation within the mitochondrial compartment. Fourth, mitochondrial protein extracts prepared from human cells overexpressing antisense DNA ligase III mRNA possessed substantially less DNA ligase activity than did mitochondrial extracts prepared from control cells. DNA end-joining activity was also substantially reduced in extracts prepared from antisense mRNA-expressing cells. From these results, we conclude that the human DNA ligase III gene encodes both nuclear and mitochondrial enzymes. DNA ligase plays a central role in DNA replication, recombination, and DNA repair. Thus, identification of a mitochondrial form of this enzyme provides a tool with which to dissect mammalian mitochondrial genome dynamics.     

CONTROL OF MITOCHONDRIAL TURNOVER UNDER THE INFLUENCE OF THYROID HORMONE

The effect of thyroid hormone on the turnover of mitochondrial DNA and protein was studied in rat heart and liver. Changes in turnover were observed in both thyroidectomized and normal rats following administration of thyroid hormone. In heart and liver the turnover of mitochondrial DNA and protein was slower in thyroidectomized rats than in normal rats. The turnover of mitochondrial DNA and protein was affected similarly following the administration of thyroid hormone, suggesting that mechanisms which control turnover of mitochondrial constituents may be predicated upon a major part of the mitochondrion. In heart a decreased rate of degradation contributes to the increase in total mitochondrial protein. Mitochondrial DNA, labeled before administration of thyroid hormone, turns over, after the start of thyroid hormone administration, at a different rate from that in newly synthesized DNA. The different turnover rates suggest that in liver the pre-existing population of mitochondria is being replaced by another population synthesized under new physiological conditions.     

Mechanism for generation of hydrogen peroxide and change of mitochondrial membrane potential during rotenone-induced apoptosis

Rotenone, an inhibitor of NADH dehydrogenase complex, is a naturally occurring insecticide, which is capable of inducing apoptosis. Rotenone-induced apoptosis is considered to contribute to its anticancer effect and the etiology of Parkinson's disease (PD). We demonstrated that rotenone induced internucleosomal DNA fragmentation, DNA ladder formation, in human cultured cells, HL-60 (promyelocytic leukemia) and BJAB cells (B-cell lymphoma). Flow cytometry showed that rotenone induced H2O2 generation, followed by significant changes in the mitochondrial membrane potential (DeltaPsim). Caspase-3 activity increased in HL-60 cells in a time-dependent manner. These apoptotic events were delayed in HP100 cells, an H2O2-resistant clone of HL-60, confirming the involvement of H2O2 in apoptosis. Expression of anti-apoptotic protein, Bcl-2, in BJAB cells drastically inhibited DeltaPsim change and DNA ladder formation but not H2O2 generation, confirming the participation of mitochondrial dysfunction in apoptosis. NAD(P)H oxidase inhibitors prevented H2O2 generation and DNA ladder formation. These results suggest that rotenone induces O2(-)-derived H2O2 generation through inhibition of NADH dehydrogenase complex and/or activation of NAD(P)H oxidase, and H2O2 generation causes the disruption of mitochondrial membrane in rotenone-induced apoptosis.