Showing papers on "Mitochondrial DNA replication published in 2007"

Mitochondrial DNA replication during differentiation of murine embryonic stem cells.

Abstract: Oxidative phosphorylation (OXPHOS), the intracellular process that generates the majority of the ATP of a cell through the electron-transfer chain, is highly dependent on proteins encoded by the mitochondrial genome (mtDNA). MtDNA replication is regulated by the nuclear-encoded mitochondrial transcription factor A (TFAM) and the mitochondrial-specific DNA polymerase gamma, which consists of a catalytic (POLG) and an accessory (POLG2) subunit. Differentiation of pluripotent embryonic stem cells (ESCs) into specific cell types requires expansion of discrete populations of mitochondria and mtDNA replication to meet the specific metabolic requirements of the cell. We determined by real-time PCR that expression of pluripotent markers is reduced before the upregulation of Polg , Polg2 and Tfam in spontaneously differentiating R1 murine (m)ESCs, along with transient increases in mtDNA copy number. In D3 mESCs, the initial transient increase did not take place. However, precursors of neuronal and cardiomyocyte differentiation were positive for both POLG and TFAM. Similar-stage ESCs also showed active mtDNA replication, identified by 5-bromo-2′-deoxy-uridine labelling, as mtDNA copy number increased. Retinoic-acid-induced differentiation resulted in more consistent patterns of replication and upregulation of Polg , Polg2 and Tfam , whereas siRNA knockdown demonstrated that steady-state expression of POLG is essential for maintaining pluripotency.

Plant mitochondrial recombination surveillance requires unusual RecA and MutS homologs.

Abstract: For >20 years, the enigmatic behavior of plant mitochondrial genomes has been well described but not well understood. Chimeric genes appear, and occasionally are differentially replicated or expressed, with significant effects on plant phenotype, most notably on male fertility, yet the mechanisms of DNA replication, chimera formation, and recombination have remained elusive. Using mutations in two important genes of mitochondrial DNA metabolism, we have observed reproducible asymmetric recombination events occurring at specific locations in the mitochondrial genome. Based on these experiments and existing models of double-strand break repair, we propose a model for plant mitochondrial DNA replication, chimeric gene formation, and the illegitimate recombination events that lead to stoichiometric changes. We also address the physiological and developmental effects of aberrant events in mitochondrial genome maintenance, showing that mitochondrial genome rearrangements, when controlled, influence plant reproduction, but when uncontrolled, lead to aberrant growth phenotypes and dramatic reduction of the cell cycle.

Mitochondrial DNA replication during differentiation of murine embryonic stem cells

Abstract: 1. 1. Facucho-Oliveira J. M., 2. et al. 2007. J. Cell Sci. doi:10.1242/jcs.016972 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft.jtitle%253DJ.%2BCell%2BSci.%26rft_id%253Dinfo%253Adoi%252F10.1242%252Fjcs.016972%26rft_id%253Dinfo%253Apmid%252F17971411%

Regulated Mitochondrial DNA Replication During Oocyte Maturation Is Essential for Successful Porcine Embryonic Development

Abstract: Cellular ATP is mainly generated through mitochondrial oxidative phosphorylation, which is dependent on mitochondrial DNA (mtDNA). We have previously demonstrated the importance of oocyte mtDNA for porcine and human fertilization. However, the role of nuclear-encoded mitochondrial replication factors during oocyte and embryo development is not yet understood. We have analyzed two key factors, mitochondrial transcription factor A (TFAM) and polymerase gamma (POLG), to determine their role in oocyte and early embryo development. Competent and incompetent oocytes, as determined by brilliant cresyl blue (BCB) dye, were assessed intermittently during the maturation process for TFAM and POLG mRNA using real-time RT-PCR, for TFAM and POLG protein using immunocytochemistry, and for mtDNA copy number using real-time PCR. Analysis was also carried out following treatment of maturing oocytes with the mtDNA replication inhibitor, 2',3'-dideoxycytidine (ddC). Following in vitro fertilization, preimplantation embryos were also analyzed. Despite increased levels of TFAM and POLG mRNA and protein at the four-cell stage, no increase in mtDNA copy number was observed in early preimplantation development. To compensate for this, mtDNA appeared to be replicated during oocyte maturation. However, significant differences in nuclear-encoded regulatory protein expression were observed between BCB(+) and BCB(-) oocytes and between untreated oocytes and those treated with ddC. These changes resulted in delayed mtDNA replication, which correlated to reduced fertilization and embryonic development. We therefore conclude that adherence to the regulation of the timing of mtDNA replication during oocyte maturation is essential for successful embryonic development.

Expression of catalytic mutants of the mtDNA helicase Twinkle and polymerase POLG causes distinct replication stalling phenotypes

Abstract: The mechanism of mitochondrial DNA replication is a subject of intense debate. One model proposes a strand-asynchronous replication in which both strands of the circular genome are replicated semi-independently while the other model proposes both a bidirectional coupled leading- and lagging-strand synthesis mode and a unidirectional mode in which the lagging-strand is initially laid-down as RNA by an unknown mechanism (RITOLS mode). Both the strand-asynchronous and RITOLS model have in common a delayed synthesis of the DNA-lagging strand. Mitochondrial DNA is replicated by a limited set of proteins including DNA polymerase gamma (POLG) and the helicase Twinkle. Here, we report the effects of expression of various catalytically deficient mutants of POLG1 and Twinkle in human cell culture. Both groups of mutants reduced mitochondrial DNA copy number by severe replication stalling. However, the analysis showed that while induction of POLG1 mutants still displayed delayed lagging-strand synthesis, Twinkle-induced stalling resulted in maturated, essentially fully double-stranded DNA intermediates. In the latter case, limited inhibition of POLG with dideoxycytidine restored the delay between leading- and lagging-strand synthesis. The observed cause-effect relationship suggests that Twinkle-induced stalling increases lagging-strand initiation events and/or maturation mimicking conventional strand-coupled replication.

Impact of assisted reproductive technologies: a mitochondrial perspective of cytoplasmic transplantation.

Abstract: Many of the assisted reproductive techniques associated with maternal aging, disease states, or implantation failure aim to correct poor developmental capacity. These techniques are highly invasive and require the exchange of nuclear or cytoplasmic material from a donor oocyte to compensate for deficiencies inherent in the affected individual. These techniques are based on the assumption that the cytoplasm of the donor oocyte can effectively substitute the necessary component(s) to enable development to proceed. Several studies have attempted to inject cytoplasm from "normal" (young) donors, into aged eggs, again assuming that beneficial components of the cytoplasm are transferred to restore developmental capacity. These invasive assisted reproduction technology (ART) procedures aim to eliminate chromosomal abnormalities, improve the quality of oocytes deficient in some important cytoplasmic factors necessary for maturation and/or subsequent development, and eliminate maternally inherited diseases (particularly mitochondrial myopathies). However, in order to develop such ART, understanding the processes involving mitochondrial DNA replication and transcription is imperative, as asynchrony between mitochondrial and nuclear genomes may cause problems in mitochondrial function, localization, and biogenesis.

Zidovudine inhibits thymidine phosphorylation in the isolated perfused rat heart.

Abstract: Zidovudine (AZT; 3′-azido-3′-deoxythymidine), a thymidine analog, has been a staple of highly active antiretroviral therapy. It is phosphorylated in the host to the triphosphate and functions by inhibiting the viral reverse transcriptase. However, long-term use of AZT is linked to various tissue toxicities, including cardiomyopathy. These toxicities are associated with mitochondrial DNA depletion, which is hypothesized to be caused by AZT triphosphate inhibition of mitochondrial DNA polymerase γ. In previous work with isolated heart mitochondria, we demonstrated that AZT phosphorylation beyond the monophosphate was not detected and that AZT itself was a potent inhibitor of thymidine phosphorylation. This suggests an alternative hypothesis in which depletion of the TTP pool may limit mitochondrial DNA replication. The present work extends these studies to the whole cell by investigating the metabolism of thymidine and AZT in the intact isolated perfused rat heart. [3H]thymidine is converted to [3H]TTP in a time- and concentration-dependent manner. The level of [3H]TMP is low, suggesting that the reaction catalyzed by thymidine kinase is the rate-limiting step in phosphorylation. [3H]AZT is converted in a time- and concentration-dependent manner to AZT monophosphate, the only phosphorylated product detected after 3 h of perfusion. Both compounds display negative cooperativity, similar to the observations with cloned and purified mitochondrial thymidine kinase 2. The presence of AZT in the perfusate inhibits the phosphorylation of [3H]thymidine with a 50% inhibitory concentration of 24 ± 4 μM. These data support the hypothesis that AZT-induced mitochondrial cardiotoxicity may be caused by a limiting pool of TTP that lowers mitochondrial DNA replication.

Maintaining precursor pools for mitochondrial DNA replication

Abstract: Among the human diseases that result from abnormalities in mitochondrial genome stability or maintenance are several that result from mutations affecting enzymes of deoxyribonucleoside triphosphate (dNTP) metabolism. In addition, it is evident that the toxicity of antiviral nucleoside analogs is determined in part by the extent to which their intracellular conversion to dNTP analogs occurs within the mitochondrion. Finally, recent work from this laboratory has shown considerable variation among different mammalian tissues with respect to mitochondrial dNTP pool sizes and has suggested that natural asymmetries in mitochondrial dNTP concentrations may contribute to the high rates at which the mitochondrial genome undergoes mutation. These factors suggest that much more information is needed about maintenance and regulation of dNTP pools within mammalian mitochondria. This review summarizes our current understanding and suggests directions for future research.—Mathews, C. K., Song, S. Maintaining precursor p...

Mechanisms of antiretroviral therapy-induced mitochondrial dysfunction.

Abstract: PURPOSE OF REVIEW To discuss novel developments related to the mechanisms of antiretroviral therapy-related mitochondrial toxicity, describe some apparent paradoxes in the current understanding of this field, and present questions that should be addressed by future research. RECENT FINDINGS The early polymerase gamma hypothesis states that nucleoside reverse transcriptase inhibitors can inhibit mitochondrial DNA replication and cause mitochondrial toxicity through mtDNA depletion. This mechanism is supported by a large body of evidence. Clinical manifestations of mitochondrial dysfunction are not always associated with mtDNA depletion. Increased mtDNA levels after nucleoside reverse transcriptase inhibitor exposure, as well as seemingly severe mtDNA depletion in individuals who show no clinical toxicity, have been reported. These and other observations suggest that additional mechanisms are involved in antiretroviral therapy toxicity, a notion supported by recent studies. Individuals given the same antiretroviral regimen can differ vastly with respect to the development of mitochondrial toxicity symptoms, reflecting interindividual variability. Some factors that may modulate this variability will be discussed. SUMMARY Mitochondrial toxicity induced by nucleoside reverse transcriptase inhibitors and their metabolic intermediates is probably mediated through many direct and indirect mechanisms. Depending on the mechanisms at play, the long-term health consequences of this toxicity may vary.

Mitochondrial DNA replication in pre-implantation embryonic development

Abstract: All eukaryotic cells possess mitochondrial DNA (mtDNA), which is maternally inherited through the oocyte, its replication being regulated by nuclear-encoded replication factors. It was hypothesised that mtDNA replication is highly regulated in oocytes, pre-implantation embryos and embryonic stem cells (ESCs) and that this may be disrupted following nuclear transfer (NT). MtDNA copy number decreased between 2-cell and 8-cell staged porcine embryos and increased between the morula and expanded blastocyst stages, coinciding with increased expression of mtDNA replication factors. Competent porcine oocytes replicated their mtDNA prior to and during in vitro maturation to produce and maintain the 100000 mtDNA copies required for fertilisation. Those oocytes in which mtDNA replication was delayed had reduced developmental ability. Expression of pluripotency-associated genes decreased as murine ESCs differentiated into embryoid bodies, although expression of mtDNA replication factors did not increase until the stage equivalent to organogenesis. Cross-species NT embryos in which the donor cell-derived mtDNA was replicated produced decreased developmental outcomes compared to those in which no mtDNA replication took place. Disruption of the strict regulation of mtDNA replication that occurs during early embryogenesis, as is likely following NT, may therefore contribute to the reduced developmental ability of embryos produced using such techniques.