Anticancer DNA intercalators cause p53-dependent mitochondrial DNA nucleoid re-modelling.
ABSTRACT: Many anticancer drugs, such as doxorubicin (DXR), intercalate into nuclear DNA of cancer cells, thereby inhibiting their growth. However, it is not well understood how such drugs interact with mitochondrial DNA (mtDNA). Using cell and molecular studies of cultured cells, we show that DXR and other DNA intercalators, such as ethidium bromide, can rapidly intercalate into mtDNA within living cells, causing aggregation of mtDNA nucleoids and altering the distribution of nucleoid proteins. Remodelled nucleoids excluded DXR and maintained mtDNA synthesis, whereas non-remodelled nucleoids became heavily intercalated with DXR, which inhibited their replication, thus leading to mtDNA depletion. Remodelling was accompanied by extensive mitochondrial elongation or interconnection, and was suppressed in cells lacking mitofusin 1 and optic atrophy 1 (OPA1), the key proteins for mitochondrial fusion. In contrast, remodelling was significantly increased by p53 or ataxia telangiectasia mutated inhibition (ATM), indicating a link between nucleoid dynamics and the genomic DNA damage response. Collectively, our results show that DNA intercalators can trigger a common mitochondrial response, which likely contributes to the marked clinical toxicity associated with these drugs.
Project description:Mammalian mtDNA is packaged in DNA-protein complexes denoted mitochondrial nucleoids. The organization of the nucleoid is a very fundamental question in mitochondrial biology and will determine tissue segregation and transmission of mtDNA. We have used a combination of stimulated emission depletion microscopy, enabling a resolution well below the diffraction barrier, and molecular biology to study nucleoids in a panel of mammalian tissue culture cells. We report that the nucleoids labeled with antibodies against DNA, mitochondrial transcription factor A (TFAM), or incorporated BrdU, have a defined, uniform mean size of ?100 nm in mammals. Interestingly, the nucleoid frequently contains only a single copy of mtDNA (average ?1.4 mtDNA molecules per nucleoid). Furthermore, we show by molecular modeling and volume calculations that TFAM is a main constituent of the nucleoid, besides mtDNA. These fundamental insights into the organization of mtDNA have broad implications for understanding mitochondrial dysfunction in disease and aging.
Project description:Mitochondrial DNA (mtDNA) is organized in nucleoprotein complexes called mitochondrial nucleoids (mt-nucleoids), which are critical units of mtDNA replication and transmission. In humans, several hundreds of mt-nucleoids exist in a cell. However, how numerous mt-nucleoids are maintained during the cell cycle remains elusive, because cell cycle synchronization procedures affect mtDNA replication. Here, we analyzed regulation of the maintenance of mt-nucleoids in the cell cycle, using a fluorescent cell cycle indicator, Fucci2. Live imaging of mt-nucleoids with higher temporal resolution showed frequent attachment and detachment of mt-nucleoids throughout the cell cycle. TFAM, an mtDNA packaging protein, was involved in the regulation of this dynamic process, which was important for maintaining proper mt-nucleoid number. Both an increase in mt-nucleoid number and activation of mtDNA replication occurred during S phase. To increase mt-nucleoid number, mtDNA replication, but not nuclear DNA replication, was necessary. We propose that these dynamic and regulatory processes in the cell cycle maintain several hundred mt-nucleoids in proliferating cells.
Project description:Mitochondrial DNA (mtDNA) is organized in protein-DNA macrocomplexes called nucleoids. Average nucleoids contain 2-8 mtDNA molecules, which are organized by the histone-like mitochondrial transcription factor A. Besides well-characterized constituents, such as single-stranded binding protein or polymerase gamma (Pol gamma), various other proteins with ill-defined functions have been identified. We report for the first time that mammalian nucleoids contain essential enzymes of an integral antioxidant system. Intact nucleoids were isolated with sucrose density gradients from rat and bovine heart as well as human Jurkat cells. Manganese superoxide dismutase (SOD2) was detected by Western blot in the nucleoid fractions. DNA, mitochondrial glutathione peroxidase (GPx1), and Pol gamma were coimmunoprecipitated with SOD2 from nucleoid fractions, which suggests that an antioxidant system composed of SOD2 and GPx1 are integral constituents of nucleoids. Interestingly, in cultured bovine endothelial cells the association of SOD2 with mtDNA was absent. Using a sandwich filter-binding assay, direct association of SOD2 by salt-sensitive ionic forces with a chemically synthesized mtDNA fragment was demonstrated. Increasing salt concentrations during nucleoid isolation on sucrose density gradients disrupted the association of SOD2 with mitochondrial nucleoids. Our biochemical data reveal that nucleoids contain an integral antioxidant system that may protect mtDNA from superoxide-induced oxidative damage.
Project description:Mitochondrial DNA (mtDNA) is packaged into DNA-protein assemblies called nucleoids, but the mode of mtDNA propagation via the nucleoid remains controversial. Two mechanisms have been proposed: nucleoids may consistently maintain their mtDNA content faithfully, or nucleoids may exchange mtDNAs dynamically. To test these models directly, two cell lines were fused, each homoplasmic for a partially deleted mtDNA in which the deletions were nonoverlapping and each deficient in mitochondrial protein synthesis, thus allowing the first unequivocal visualization of two mtDNAs at the nucleoid level. The two mtDNAs transcomplemented to restore mitochondrial protein synthesis but were consistently maintained in discrete nucleoids that did not intermix stably. These results indicate that mitochondrial nucleoids tightly regulate their genetic content rather than freely exchanging mtDNAs. This genetic autonomy provides a molecular mechanism to explain patterns of mitochondrial genetic inheritance, in addition to facilitating therapeutic methods to eliminate deleterious mtDNA mutations.
Project description:Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.
Project description:Mitochondrial DNA (mtDNA) is packaged into DNA-protein complexes called nucleoids, which are distributed as many small foci in mitochondria. Nucleoids are crucial for the biogenesis and function of mtDNA. Here, using a yeast genetic screen for components that control nucleoid distribution and size, we identify Fcj1 and Mos1, two evolutionarily conserved mitochondrial proteins that maintain the connection between the cristae and boundary membranes. These two proteins are also important for establishing tubular morphology of mitochondria, as mitochondria lacking Fcj1 and Mos1 form lamellar sheets. We find that nucleoids aggregate, increase in size, and decrease in number in fcj1 and mos1 cells. In addition, Fcj1 form punctate structures and localized adjacent to nucleoids. Moreover, connecting mitochondria by deleting the DNM1 gene required for organelle division enhances aggregation of mtDNA nucleoids in fcj1 and mos1 cells, whereas single deletion of DNM1 does not affect nucleoids. Conversely, deleting F1Fo-ATP synthase dimerization factors generates concentric ring-like cristae, restores tubular mitochondrial morphology, and suppresses nucleoid aggregation in these mutants. Our findings suggest an unexpected role of Fcj1-Mos1 and organelle division in maintaining the distribution and size of mtDNA nucleoids.
Project description:Mitochondrial DNA is organized as a nucleoprotein complex called the nucleoid. Its major protein components have been identified in different organisms, but it is yet unknown whether nucleoids undergo any form of remodeling. Using an in organello ChIP-on-chip assay, we demonstrate that the DNA-bending protein Abf2 binds to most of the mitochondrial genome with a preference for GC-rich gene sequences. Thus, Abf2 is a bona fide mitochondrial DNA-packaging protein in vivo. Nucleoids form a more open structure under respiring growth conditions in which the ratio of Abf2 to mitochondrial DNA is decreased. Bifunctional nucleoid proteins Hsp60 and Ilv5 are recruited to nucleoids during glucose repression and amino-acid starvation, respectively. Thus, mitochondrial nucleoids in yeast are dynamic structures that are remodeled in response to metabolic cues. A mutant form of Hsp60 that causes mtDNA instability has altered submitochondrial localization, which suggests that nucleoid remodeling is essential for the maintenance of mitochondrial genome.
Project description:Mitochondrial DNA (mtDNA) is organized in discrete protein-DNA complexes, nucleoids, that are usually considered to be mitochondrial-inner-membrane associated. Here we addressed the association of replication factors with nucleoids and show that endogenous mtDNA helicase Twinkle and single-stranded DNA-binding protein, mtSSB, co-localize only with a subset of nucleoids. Using nucleotide analogs to identify replicating mtDNA in situ, the fraction of label-positive nucleoids that is Twinkle/mtSSB positive, is highest with the shortest labeling-pulse. In addition, the recruitment of mtSSB is shown to be Twinkle dependent. These proteins thus transiently associate with mtDNA in an ordered manner to facilitate replication. To understand the nature of mtDNA replication complexes, we examined nucleoid protein membrane association and show that endogenous Twinkle is firmly membrane associated even in the absence of mtDNA, whereas mtSSB and other nucleoid-associated proteins are found in both membrane-bound and soluble fractions. Likewise, a substantial amount of mtDNA is found as soluble or loosely membrane bound. We show that, by manipulation of Twinkle levels, mtDNA membrane association is partially dependent on Twinkle. Our results thus show that Twinkle recruits or is assembled with mtDNA at the inner membrane to form a replication platform and amount to the first clear demonstration that nucleoids are dynamic both in composition and concurrent activity.
Project description:Mitochondrial DNA (mtDNA) is packaged by association with specific proteins in compact DNA-protein complexes named mitochondrial nucleoids (mt-nucleoids). The budding yeast Saccharomyces cerevisiae is able to grow either aerobically or anaerobically. Due to this characteristic, S. cerevisiae has been extensively used as a model organism to study genetics, morphology and biochemistry of mitochondria for a long time. Mitochondria of S. cerevisiae frequently fuse and divide, and perform dynamic morphological changes depending on the culture conditions and the stage of life cycle of the yeast cells. The mt-nucleoids also dynamically change their morphology, accompanying morphological changes of mitochondria. The mt-nucleoids have been isolated morphologically intact and functional analyses of mt-nucleoid proteins have been extensively performed. These studies have revealed that the functions of mt-nucleoid proteins are essential for maintenance of mtDNA. The aims of this review are to summarize the history on the research of yeast mt-nucleoids as well as recent findings on the organization of the mt-nucleoids and mitochondrial dynamics.
Project description:Mitochondrial DNA (mtDNA) is packed into highly organized structures called mitochondrial nucleoids (mt-nucleoids). To understand the organization of mtDNA and the overall regulation of its genetic activity within the mt-nucleoids, we identified and characterized a novel mtDNA packaging protein, termed Glom (a protein inducing agglomeration of mitochondrial chromosome), from highly condensed mt-nucleoids of the true slime mold, Physarum polycephalum. This protein could bind to the entire mtDNA and package mtDNA into a highly condensed state in vitro. Immunostaining analysis showed that Glom specifically localized throughout the mt-nucleoid. Deduced amino acid sequence revealed that Glom has a lysine-rich region with proline-rich domain in the N-terminal half and two HMG boxes in C-terminal half. Deletion analysis of Glom revealed that the lysine-rich region was sufficient for the intense mtDNA condensation in vitro. When the recombinant Glom proteins containing the lysine-rich region were expressed in Escherichia coli, the condensed nucleoid structures were observed in E. coli. Such in vivo condensation did not interfere with transcription or replication of E. coli chromosome and the proline-rich domain was essential to keep those genetic activities. The expression of Glom also complemented the E. coli mutant lacking the bacterial histone-like protein HU and the HMG-boxes region of Glom was important for the complementation. Our results suggest that Glom is a new mitochondrial histone-like protein having a property to cause intense DNA condensation without suppressing DNA functions.