Mitochondrial transcription in mammalian cells.

As a consequence of recent discoveries of intimate involvement of mitochondria with key cellular processes, there has been a resurgence of interest in all aspects of mitochondrial biology, including the intricate mechanisms of mitochondrial DNA maintenance and expression. Despite four decades of research, there remains a lot to be learned about the processes that enable transcription of genetic information from mitochondrial DNA to RNA, as well as their regulation. These processes are vitally important, as evidenced by the lethality of inactivating the central components of mitochondrial transcription machinery. Here, we review the current understanding of mitochondrial transcription and its regulation in mammalian cells. We also discuss key theories in the field and highlight controversial subjects and future directions as we see them.

The efficiency of the translesion synthesis across abasic sites by mitochondrial DNA polymerase is low in mitochondria of 3T3 cells.

Translesion synthesis by specialized DNA polymerases is an important strategy for mitigating DNA damage that cannot be otherwise repaired either due to the chemical nature of the lesion. Apurinic/Apyrimidinic (abasic, AP) sites represent a block to both transcription and replication, and are normally repaired by the base excision repair (BER) pathway. However, when the number of abasic sites exceeds BER capacity, mitochondrial DNA is targeted for degradation. Here, we used two uracil-N-glycosylase (UNG1) mutants, Y147A or N204D, to generate AP sites directly in the mtDNA of NIH3T3 cells in vivo at sites normally occupied by T or C residues, respectively, and to study repair of these lesions in their native context. We conclude that mitochondrial DNA polymerase γ (Pol γ) is capable of translesion synthesis across AP sites in mitochondria of the NIH3T3 cells, and obeys the A-rule. However, in our system, base excision repair (BER) and mtDNA degradation occur more frequently than translesion bypass of AP sites.

The "fast" and the "slow" modes of mitochondrial DNA degradation.

In a living cell, oxidative stress resulting from an external or internal insult can result in mitochondrial DNA (mtDNA) damage and degradation. Here, we show that in HeLa cells, mtDNA can withstand relatively high levels of extracellular oxidant H2O2 before it is damaged to a point of degradation, and that mtDNA levels in these cells quickly recover after removal of the stressor. In contrast, mtDNA degradation in mouse fibroblast cells is induced at eight-fold lower concentrations of H2O2, and restoration of the lost mtDNA proceeds much slower. Importantly, mtDNA levels in HeLa cells continue to decline even after withdrawal of the stressor thus marking the "slow" mode of mtDNA degradation. Conversely, in mouse fibroblasts maximal loss of mtDNA is achieved during treatment, and is already detectable at 5 min after exposure, indicating the "fast" mode. These differences may modulate susceptibility to oxidative stress of those organs, which consist of multiple cell types.

Mitochondrial DNA: A disposable genome?

In mammalian cells, mitochondria are the only organelles besides the nucleus that house genomic DNA. The mammalian mitochondrial genome is represented by prokaryotic-type, circular, highly compacted DNA molecules. Today, more than a half-century after their discovery, the biology of these small and redundant molecules remains much less understood than that of their nuclear counterparts. One peculiarity of the mitochondrial genome that emerged in recent years is its disposable nature, as evidenced by cells abandoning a fraction of their mitochondrial DNA (mtDNA) in response to various stimuli with little or no physiological consequence. Here, we review some recent developments in the field of mtDNA biology and discuss emerging questions on the disposability and indispensability of mtDNA.

Aging: A mitochondrial DNA perspective, critical analysis and an update.

The mitochondrial theory of aging, a mainstream theory of aging which once included accumulation of mitochondrial DNA (mtDNA) damage by reactive oxygen species (ROS) as its cornerstone, has been increasingly losing ground and is undergoing extensive revision due to its inability to explain a growing body of emerging data. Concurrently, the notion of the central role for mtDNA in the aging process is being met with increased skepticism. Our progress in understanding the processes of mtDNA maintenance, repair, damage, and degradation in response to damage has largely refuted the view of mtDNA as being particularly susceptible to ROS-mediated mutagenesis due to its lack of "protective" histones and reduced complement of available DNA repair pathways. Recent research on mitochondrial ROS production has led to the appreciation that mitochondria, even in vitro, produce much less ROS than previously thought, automatically leading to a decreased expectation of physiologically achievable levels of mtDNA damage. New evidence suggests that both experimentally induced oxidative stress and radiation therapy result in very low levels of mtDNA mutagenesis. Recent advances provide evidence against the existence of the "vicious" cycle of mtDNA damage and ROS production. Meta-studies reveal no longevity benefit of increased antioxidant defenses. Simultaneously, exciting new observations from both comparative biology and experimental systems indicate that increased ROS production and oxidative damage to cellular macromolecules, including mtDNA, can be associated with extended longevity. A novel paradigm suggests that increased ROS production in aging may be the result of adaptive signaling rather than a detrimental byproduct of normal respiration that drives aging. Here, we review issues pertaining to the role of mtDNA in aging.

Mitochondrial DNA ligase is dispensable for the viability of cultured cells but essential for mtDNA maintenance.

Multiple lines of evidence support the notion that DNA ligase III (LIG3), the only DNA ligase found in mitochondria, is essential for viability in both whole organisms and in cultured cells. Previous attempts to generate cells devoid of mitochondrial DNA ligase failed. Here, we report, for the first time, the derivation of viable LIG3-deficient mouse embryonic fibroblasts. These cells lack mtDNA and are auxotrophic for uridine and pyruvate, which may explain the apparent lethality of the Lig3 knock-out observed in cultured cells in previous studies. Cells with severely reduced expression of LIG3 maintain normal mtDNA copy number and respiration but show reduced viability in the face of alkylating and oxidative damage, increased mtDNA degradation in response to oxidative damage, and slow recovery from mtDNA depletion. Our findings clarify the cellular role of LIG3 and establish that the loss of viability in LIG3-deficient cells is conditional and secondary to the ρ(0) phenotype.

Persistent damage induces mitochondrial DNA degradation.

Considerable progress has been made recently toward understanding the processes of mitochondrial DNA (mtDNA) damage and repair. However, a paucity of information still exists regarding the physiological effects of persistent mtDNA damage. This is due, in part, to experimental difficulties associated with targeting mtDNA for damage, while sparing nuclear DNA. Here, we characterize two systems designed for targeted mtDNA damage based on the inducible (Tet-ON) mitochondrial expression of the bacterial enzyme, exonuclease III, and the human enzyme, uracil-N-glyosylase containing the Y147A mutation. In both systems, damage was accompanied by degradation of mtDNA, which was detectable by 6h after induction of mutant uracil-N-glycosylase and by 12h after induction of exoIII. Unexpectedly, increases in the steady-state levels of single-strand lesions, which led to degradation, were small in absolute terms indicating that both abasic sites and single-strand gaps may be poorly tolerated in mtDNA. mtDNA degradation was accompanied by the loss of expression of mtDNA-encoded COX2. After withdrawal of the inducer, recovery from mtDNA depletion occurred faster in the system expressing exonuclease III, but in both systems reduced mtDNA levels persisted longer than 144h after doxycycline withdrawal. mtDNA degradation was followed by reduction and loss of respiration, decreased membrane potential, reduced cell viability, reduced intrinsic reactive oxygen species production, slowed proliferation, and changes in mitochondrial morphology (fragmentation of the mitochondrial network, rounding and "foaming" of the mitochondria). The mutagenic effects of abasic sites in mtDNA were low, which indicates that damaged mtDNA molecules may be degraded if not rapidly repaired. This study establishes, for the first time, that mtDNA degradation can be a direct and immediate consequence of persistent mtDNA damage and that increased ROS production is not an invariant consequence of mtDNA damage.

The approaches for manipulating mitochondrial proteome.

Over the past decade a large volume of research data has accumulated which has established a fundamental role for mitochondria in normal cellular functioning, as well as in various pathologies. Mitochondria play a pivotal role in metabolism and energy production, and are one of the key players involved in programmed cell death. On the other hand, mitochondrial dysfunction is implicated, directly or indirectly in numerous pathological conditions including inherited mitochondrial disorders, diabetes, cardiovascular and neurodegenerative diseases, and a variety of malignancies. The ability to modulate mitochondrial function by altering the diverse protein component of this organelle may be of great value for developing future therapeutic interventions. This review will discuss approaches used to introduce proteins into mitochondria. One group of methods utilizes strategies aimed at expressing proteins from genes in the nucleus. These include overexpression of nuclear-encoded mitochondrial proteins, allotopic expression, which is the re-coding and relocation of mitochondrial genes to the nucleus for expression and subsequent delivery of their gene products to mitochondria, and xenotopic expression, which is the nuclear expression of genes coding electron transport chain components from distant species, for delivery of their products to mammalian mitochondria. Additionally, antigenomic and progenomic strategies which focus on expression of mitochondrially targeted nuclear proteins involved in the maintenance of mtDNA will be discussed. The second group of methods considered will focus on attempts to use purified proteins for mitochondrial delivery. Special consideration has been given to the complexities involved in targeting exogenous proteins to mitochondria.

A retro-lentiviral system for doxycycline-inducible gene expression and gene knockdown in cells with limited proliferative capacity.

Currently, there is no reliable system for regulated gene expression and regulated gene knockdown in cells with finite lifespan. In this manuscript, we describe a vector system, consisting of a retrovirus for the delivery of rtTA, and a lentivirus for the delivery of either a transgene or a miR-shRNA for the modification of primary cells. Primary rat pulmonary microvascular endothelial cells (PMVEC) modified by these vectors for the inducible expression of Gaussia luciferase or DsRed Express demonstrated greater than 100-fold induction of the transgene expression with doxycycline. The system works reliably in both sequential and simultaneous infection modes, with about 95% of the sells selected with two antibiotics being inducible in each mode. The lentiviral vector for gene knockdown allows for the direct cloning of shRNA oligos using alpha-complementation, and for the monitoring of induction of RNA interference with fluorescent reporter, mCherry. The gene knockdown vector was validated by knocking down beta-actin expression in PMVECs, with two of the four constructs showing 59 and 75% knockdown, respectively, compared to uninduced controls. The vectors described here were successfully used for the modification of various primary and established cell lines for regulated gene expression and regulated knockdown.

Mitochondrial DNA damage initiates a cell cycle arrest by a Chk2-associated mechanism in mammalian cells.

Previous work from our laboratory has focused on mitochondrial DNA (mtDNA) repair and cellular viability. However, other events occur prior to the initiation of apoptosis in cells. Because of the importance of mtDNA in ATP production and of ATP in fuel cell cycle progression, we asked whether mtDNA damage was an upstream signal leading to cell cycle arrest. Using quantitative alkaline Southern blot technology, we found that exposure to menadione produced detectable mtDNA damage in HeLa cells that correlated with an S phase cell cycle arrest. To determine whether mtDNA damage was causatively linked to the observed cell cycle arrest, experiments were performed utilizing a MTS-hOGG1-Tat fusion protein to target the hOGG1 repair enzyme to mitochondria and enhance mtDNA repair. The results revealed that the transduction of MTS-hOGG1-Tat into HeLa cells alleviated the cell cycle block following an oxidative insult. Furthermore, mechanistic studies showed that Chk2 phosphorylation was enhanced following menadione exposure. Treatment of the HeLa cells with the hOGG1 fusion protein prior to menadione exposure resulted in an increase in the rate of Chk2 dephosphorylation. These results strongly support a direct link between mtDNA damage and cell cycle arrest.

Targeting repair proteins to the mitochondria of mammalian cells through stable transfection, transient transfection, viral transduction, and TAT-mediated protein transduction.

The mitochondrial genome represents a target for exogenous and endogenous damage. Its necessity for successful electron transport makes its repair valuable to the cell. Previous work from our lab has shown that mitochondrial DNA (mtDNA) can be repaired in mammalian cells, and the use of mitochondrial-targeted repair proteins can augment repair to enhance viability following genotoxic stress. In addition, it has also been shown that other repair enzymes that are targeted to the mitochondria can sensitize the cell to DNA damaging agents, thereby aiding the effectiveness of certain chemotherapeutic agents. The methods herein describe the development of mitochondrial-targeted proteins using plasmids or protein transduction domains. It includes the utilization of these constructs to create stably transfected cell lines, transiently transfected cell lines, viral-mediated transduction, and protein transduction domain-mediated mitochondrial protein localization. The end result will be a mammalian cell that expresses the mitochondrial-targeted protein of interest.

Mutations in the passenger polypeptide can affect its partitioning between mitochondria and cytoplasm: mutations can impair the mitochondrial import of DsRed.

In this study, we report that the partitioning between mitochondria and cytoplasm of two variants, mCherry and DsRed Express (DRE), of the red fluorescent protein, DsRed, fused to one of the six matrix targeting sequences (MTSs) can be affected by both MTS and amino acid substitutions in DsRed. Of the six MTSs tested, MTSs from superoxide dismutase and DNA polymerase gamma failed to direct mCherry, but not DRE to mitochondria. By evaluating a series of chimeras between mCherry and DRE fused to the MTS of superoxide dismutase, we attribute the differences in the mitochondrial partitioning to differences in the primary amino acid sequence of the passenger polypeptide. The impairment of mitochondrial partitioning closely parallels the number of mCherry-specific mutations, and is not specific to mutations located in any particular region of the polypeptide. These observations suggest that both MTS and the passenger polypeptide affect the efficiency of mitochondrial import and provide a rationale for the observed diversity in the primary amino acid sequences of natural MTSs.

TAT-mediated protein transduction and targeted delivery of fusion proteins into mitochondria of breast cancer cells.

The protein transduction domain (PTD) from the HIV-1 TAT protein has been widely utilized to deliver biologically active macromolecules, including full-length proteins, into a variety of cell types in vitro and in vivo. Without additional targeting signals, the intracellular localization of the proteins delivered in this fashion appears to be cytoplasmic, nuclear or, as recently reported, endosomal. In this study, we show that the presence of the mitochondrial targeting signal (MTS) from hMnSOD on the N-terminus of TAT-fusion proteins directs them into mitochondria of breast cancer cells. We generated and purified fusion proteins containing GFP (MTS-GFP-TAT) or Exonuclease III (MTS-ExoIII-TAT) from Escherichia coli. The results of Western blots of subcellular fractions and fluorescent microscopic analyses revealed efficient protein transduction and mitochondrial localization of the fusion proteins. Specific exonuclease activity was found in the mitochondrial extracts isolated from MTS-ExoIII-TAT transduced cells. This increased exonuclease activity reduced the repair of mtDNA damage following oxidative stress. This diminished mtDNA repair led to a decrease in survival of breast cancer cells. Thus, the present study demonstrates the applicability of this new approach for intramitochondrial targeting of TAT-fusion proteins capable of modulating mitochondrial function and cell survival.

alpha-complementation-enabled T7 expression vectors and their use for the expression of recombinant polypeptides for protein transduction experiments.

Over the past few years protein transduction has emerged as a powerful means for the delivery of proteins into cultured cells and into whole mice. This method is based on the ability of proteins containing protein transduction domains (PTDs), short stretches of 9-16 predominantly basic amino acids, to traverse the cytoplasmic membrane and accumulate inside cells in a time- and dose-dependent fashion. The number of PTDs, both natural and synthetic, is constantly expanding, as is the need to test newly discovered PTDs for their ability to mediate the internalization of the corresponding fusion proteins. Here we describe a strategy and methodology that can be used for the construction of vectors for the T7 RNA polymerase-driven expression of PTD fusions. The cloning in these vectors is facilitated by alpha-complementation. Also, these vectors are small in size (less than 3 kbp) and express influenza virus hemagglutinin tag as well as His tag as part of the fusion for immunological identification and purification respectively of expressed proteins.

The expression of Exonuclease III from E. coli in mitochondria of breast cancer cells diminishes mitochondrial DNA repair capacity and cell survival after oxidative stress.

The ability to sensitize cancer cells to radiation would be highly beneficial for successful cancer treatment. One mode of action for ionizing radiation is the induction of cell death through infliction of extensive oxidative damage to cellular DNA, including mitochondrial DNA (mtDNA). The ability of cells to repair mtDNA and otherwise maintain the integrity of their mitochondria is vital for protection of the cells against oxidative damage. Because efficient repair of oxidative damage in mtDNA may play a crucial role in cancer cell resistance, interference with this repair process could be an effective way to achieve a radiation sensitive phenotype in otherwise resistant cancer cells. Successful repair of DNA is achieved through a precise and highly regulated multistep process. Expression of excessive amounts of one of the repair enzymes may cause an imbalance of the whole repair system and lead to the loss of repair efficiency. To study the effects of changing mtDNA repair capacity on overall cell survival following oxidative stress, we expressed a bacterial repair enzyme, Exonuclease III (ExoIII) containing the mitochondrial targeting signal of manganese superoxide dismutase, in a human malignant breast epithelial cell line, MDA-MB-231. Following transfection, specific exonuclease activity was found in mitochondrial extracts. In order to examine the effects on repair of oxidative damage in mtDNA, cells were exposed to the enzyme xanthine oxidase and its substrate hypoxanthine. mtDNA repair was evaluated using quantitative Southern blot analysis. The results revealed that cells expressing ExoIII in mitochondria are deficient in mtDNA repair when compared with control cells that express ExoIII without MTS. This diminished mtDNA repair capacity rendered MDA-MB-231 cells more sensitive to oxidative damage, which resulted in a decrease in their long-term survival following oxidative stress.