The nucleus of eukaryotic cells is a vitally important organelle that sequesters the genetic information of the cell, and protects it with the help of two highly evolved structures, the nuclear envelope (NE) and nuclear pore complexes (NPCs). Together, these two structures mediate the bidirectional trafficking of molecules between the nucleus and cytoplasm by forming a barrier. NE transmembrane proteins (NETs) embedded in either the outer nuclear membrane (ONM) or the inner nuclear membrane (INM) play crucial roles in both nuclear structure and functions, including: genome architecture, epigenetics, transcription, splicing, DNA replication, nuclear structure, organization and positioning. Furthermore, numerous human diseases are associated with mutations and mislocalization of NETs on the NE. There are still many fundamental questions that are unresolved with NETs, but we focused on two major questions: First, the localization and transport rate of NETs, and second, the transport route taken by NETs to reach the INM. Since NETs are involved with many of the mechanisms used to maintain cellular homeostasis, it is important to quantitatively determine the spatial locations of NETs along the NE to fully understand their role in these vital processes. However, there are limited available approaches for this task, and moreover, these methods provide no information about the translocation rates of NETs between the two membranes. Furthermore, while the trafficking of soluble proteins between the cytoplasm and the nucleus has been well studied over the years, the path taken by NETs into the nucleus remains in dispute. At least four distinct models have been proposed to suggest how transmembrane proteins destined for the INM cross the NE through NPC-dependent or NPC-independent mechanisms, based on specific features found on the soluble domains of INM proteins. In order to resolve these two major questions, it is necessary to employ techniques with the capabilities to observe these dynamics at the nanoscale. Current experimental techniques are unable to break the temporal and spatial resolution barriers required to study these phenomena. Therefore, we developed and modified single-molecule techniques to answer these questions. First, to study the distribution of NETs on the NE, we developed a new single-molecule microscopy method called single-point single-molecule fluorescence recovery after photobleaching (smFRAP), which is able to provide spatial resolution <10 nm and, furthermore, provide previously unattainable information about NET translocation rates from the ONM to INM. Secondly, to examine the transport route used by NETs destined for the INM, we used a single-molecule microscopy technique previously developed in our lab called single-point edge-excitation sub-diffraction (SPEED) microscopy, which provides spatio-temporal resolution of <10 nm precision and 0.4 ms detection time. The major findings from my doctoral research work can be classified into two categories: (i) Technical developments to study NETs in vivo, and (ii) biological findings from employing these microscopy techniques. In regards to technical contributions, we created and validated of a new single-molecule microscopy method, smFRAP, to accurately determine the localization and distribution ratios of NETs on both the ONM and INM in live cells. Second, we adapted SPEED microscopy to study transmembrane protein translocation in vivo. My work has also contributed four main biological findings to the field: first, we determined the in vivo translocation rates for lamin-B receptor (LBR), a major INM protein found in the nucleus of cells. Second, we verified the existence of peripheral channels in the scaffolding of NPCs and, for the first time, directly observed the transit of INM proteins through these channels in live cells. Third, our research has elucidated the roles that both the nuclear localization signal (NLS) and intrinsically disordered (ID) domains play in INM protein transport. Finally, my work has elucidated which transport routes are used by NETs destined to localize in the INM. / Biology

The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions-ssDNA, TPP1, or TRF2 binding-was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres. / Dissertation

A neurodegeneração é um processo onde ocorre morte celular progressiva. O tráfego neuronal anterógrado e retrógado, e entre os compartimentos é essencial para a viabilidade celular. As proteínas Rabs pertencem à família de pequenas GTPases, com funções de tráfego de vesículas e organelas, para realizarem sua função as proteínas Rab podem recrutar proteínas motoras como as KIF 1B e KIF 5, responsáveis pelo transporte anterógrado mitocondrial. A associação do distúrbio do tráfego intracelular com doenças neurodegenerativas tem sido tema de estudos recentes. Com isso o objetivo do presente trabalho é analisar a expressão das proteínas Rab, bem como estudar as proteínas motoras que podem contribuir para o esclarecimento sobre os distúrbios no tráfego intracelular que antecedem a formação de agregados proteicos envolvidos em neurodegeneração. Para tanto, utilizou-se o modelo de tratamento com rotenona para indução de agregados em Ratos Lewis idosos que foram expostos a rotenona durante 4 semanas, em seguida foram avaliados os níveis de expressão das proteínas Rab no hipocampo, substância negra e locus coeruleus, por western blotting. Foram analisados também os níveis de expressão das proteínas motoras KIF1B e KIF5 antes e durante a formação de agregados proteicos, em culturas de células, de ratos Lewis neonatos, do hipocampo, substância negra e locus coeruleus tratadas com rotenona por 24 horas ou 48 horas nas concentrações de 0,1nM, 0, 3nM e 0,5nM. Foi observado diminuição dos níveis de expressão das proteínas Rab 1 nas regiões do hipocampo e locus coeruleus. Houve aumento de expressão das Rab 4,5 e 6 no hipocampo, porém na substância negra a expressão da Rab 1 aumentou e da Rab 6 diminuiu. Já no locus coeruleus in vivo a Rab 6 aumentou, mas as Rab 1, 5 e 11 diminuíram sua expressão. Já a expressão da KIF 5 aumentou com o tratamento de 0,1nM de rotenona e diminuiu após 0,5nM do xenobiótico por 48 horas in vitro, na mesma região. Na substância negra aumentaram as KIFs 1B e 5 após o tratamento com 0,5nM por 48 horas in vitro, mas diminuíram as KIF 1B e 5 após o tratamento com 0,3nM por 24 horas e KIF 5 após o tratamento com 0,1nM por 48 horas. Esses resultados permitem concluir que a expressão de proteínas importantes para o tráfego mitocondrial e de vesículas encontram-se alteradas e fazem parte dos eventos intracelulares que antecedem a neurodegeneração / Neurodegeneration is a process that leads to progressive cell death. The anterograde and retrograde neuronal traffic as well as the traffic between compartments are essential for cell viability. The Rab proteins belong to the small GTPases family with function of vesicles and organelle trafficking. Rab proteins can recruit motor proteins such as KIF 1B and KIF 5 that are responsible for anterograde mitochondrial transport. The association of intracellular traffic disturb with neurodegenerative diseases have been theme of recent studies. Thereat the objective of this study is analyze the expression of Rab and motor proteins that can contribute for the understanding about the disturb of the intracellular traffic that precedes protein aggregation involved in neurodegeneration. For this purpose it was employed the model of rotenone treatment for induction of aggregation in aged Lewis rats that were exposed to rotenone during 4 weeks in order to evaluate Rabs expression. The levels of motor proteins KIF 1B and KIF 5 expression were evaluated before and during the formation of protein aggregates in hippocampus, substantia nigra and locus coeruleus cell cultures of neonates Lewis rats, exposed to rotenone for 24 hours or 48 hours in the concentrations of 0.1nM, 0.3nM or 0.5nM. It was observed decreased levels of Rab 1 expression in hippocampus and locus coeruleus. Rabs 4,5 and 6 were increased in the hippocampus, but in the substantia nigra the expression of Rab 1 increased and Rab 6 decreased. In the locus coeruleus the Rab 6 increased, but Rabs 1, 5 and 11 decreased. The expression of KIF 5 increased after 0.1nM of rotenone and decreased after the exposure to 0.5nM of for 48 hours in cultured cell from the locus coeruleus. In the substantia nigra the KIF1B and KIF 5 increased after treatment with 0.5nM for 48 hours in vitro, but these protein decreased after treatment with 0.3nM for 24 hours in vitro, and KIF 5 after treatment with 0.1nM for 48 hours. These results allow us conclude that the expression of important proteins for the mitochondrial and vesicles traffic are altered and participate of intracellular events that precede the neurodegeneration

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 103-117). Also issued on the Internet.

A neurodegeneração é um processo onde ocorre morte celular progressiva. O tráfego neuronal anterógrado e retrógado, e entre os compartimentos é essencial para a viabilidade celular. As proteínas Rabs pertencem à família de pequenas GTPases, com funções de tráfego de vesículas e organelas, para realizarem sua função as proteínas Rab podem recrutar proteínas motoras como as KIF 1B e KIF 5, responsáveis pelo transporte anterógrado mitocondrial. A associação do distúrbio do tráfego intracelular com doenças neurodegenerativas tem sido tema de estudos recentes. Com isso o objetivo do presente trabalho é analisar a expressão das proteínas Rab, bem como estudar as proteínas motoras que podem contribuir para o esclarecimento sobre os distúrbios no tráfego intracelular que antecedem a formação de agregados proteicos envolvidos em neurodegeneração. Para tanto, utilizou-se o modelo de tratamento com rotenona para indução de agregados em Ratos Lewis idosos que foram expostos a rotenona durante 4 semanas, em seguida foram avaliados os níveis de expressão das proteínas Rab no hipocampo, substância negra e locus coeruleus, por western blotting. Foram analisados também os níveis de expressão das proteínas motoras KIF1B e KIF5 antes e durante a formação de agregados proteicos, em culturas de células, de ratos Lewis neonatos, do hipocampo, substância negra e locus coeruleus tratadas com rotenona por 24 horas ou 48 horas nas concentrações de 0,1nM, 0, 3nM e 0,5nM. Foi observado diminuição dos níveis de expressão das proteínas Rab 1 nas regiões do hipocampo e locus coeruleus. Houve aumento de expressão das Rab 4,5 e 6 no hipocampo, porém na substância negra a expressão da Rab 1 aumentou e da Rab 6 diminuiu. Já no locus coeruleus in vivo a Rab 6 aumentou, mas as Rab 1, 5 e 11 diminuíram sua expressão. Já a expressão da KIF 5 aumentou com o tratamento de 0,1nM de rotenona e diminuiu após 0,5nM do xenobiótico por 48 horas in vitro, na mesma região. Na substância negra aumentaram as KIFs 1B e 5 após o tratamento com 0,5nM por 48 horas in vitro, mas diminuíram as KIF 1B e 5 após o tratamento com 0,3nM por 24 horas e KIF 5 após o tratamento com 0,1nM por 48 horas. Esses resultados permitem concluir que a expressão de proteínas importantes para o tráfego mitocondrial e de vesículas encontram-se alteradas e fazem parte dos eventos intracelulares que antecedem a neurodegeneração / Neurodegeneration is a process that leads to progressive cell death. The anterograde and retrograde neuronal traffic as well as the traffic between compartments are essential for cell viability. The Rab proteins belong to the small GTPases family with function of vesicles and organelle trafficking. Rab proteins can recruit motor proteins such as KIF 1B and KIF 5 that are responsible for anterograde mitochondrial transport. The association of intracellular traffic disturb with neurodegenerative diseases have been theme of recent studies. Thereat the objective of this study is analyze the expression of Rab and motor proteins that can contribute for the understanding about the disturb of the intracellular traffic that precedes protein aggregation involved in neurodegeneration. For this purpose it was employed the model of rotenone treatment for induction of aggregation in aged Lewis rats that were exposed to rotenone during 4 weeks in order to evaluate Rabs expression. The levels of motor proteins KIF 1B and KIF 5 expression were evaluated before and during the formation of protein aggregates in hippocampus, substantia nigra and locus coeruleus cell cultures of neonates Lewis rats, exposed to rotenone for 24 hours or 48 hours in the concentrations of 0.1nM, 0.3nM or 0.5nM. It was observed decreased levels of Rab 1 expression in hippocampus and locus coeruleus. Rabs 4,5 and 6 were increased in the hippocampus, but in the substantia nigra the expression of Rab 1 increased and Rab 6 decreased. In the locus coeruleus the Rab 6 increased, but Rabs 1, 5 and 11 decreased. The expression of KIF 5 increased after 0.1nM of rotenone and decreased after the exposure to 0.5nM of for 48 hours in cultured cell from the locus coeruleus. In the substantia nigra the KIF1B and KIF 5 increased after treatment with 0.5nM for 48 hours in vitro, but these protein decreased after treatment with 0.3nM for 24 hours in vitro, and KIF 5 after treatment with 0.1nM for 48 hours. These results allow us conclude that the expression of important proteins for the mitochondrial and vesicles traffic are altered and participate of intracellular events that precede the neurodegeneration

Abstract Skeletal muscle cells (myofibers) are huge multinucleated cells responsible for muscle contraction and hence for the everyday movements of the joints. The structure of these voluminous cells differs greatly from that of the mononucleated cells – the characteristic features of the myofibers include dozens of peripherally located nuclei, tightly packed contractile apparatus and a sophisticatedly organized endomembrane system. The basic physiology involving myofibers is quite well known, but scarce data exist on the membrane biology of the myofibers. The purpose of this study was to examine the localization of mRNA and the site of protein synthesis in the myofibers. The characterization of the membrane dynamics in muscle cells was also performed. In this study we utilized a primary cell culture model obtained from the rat flexor digitorum brevis (FDB) muscle. Also frozen sections from the rat extensor digitorum longus muscle were used. The precursor cells of the myofibers – myoblasts and myotubes – were also utilized in some experiments. Furthermore, methods of immunohistochemistry and molecular biology were applied extensively in this study. We found that in FDB myofibers the mRNA lies just under the plasma membrane. Protein synthesis seemed to be concentrated in the vicinity of nuclei locating beneath the plasma membrane but also in interfibrillar dot-like structures. Protein products moved hundreds of micrometers away from the nuclei of origin. Moreover, there were no barriers for protein movement into the core regions of the myofibers. Movement of proteins was found to be rapid in the cytosol and in the endomembrane system, too. Interestingly, when examining exocytic trafficking we observed that ER-to-Golgi trafficking significantly differed from that of mononucleated cells. Finally, myofibers were found to be able to generate lipid bodies under stress conditions. The dynamics of lipid bodies seemed to deviate from the dynamics found in other cells types. Nowadays not much muscle research with primary myofibers is done worldwide, and therefore dilemmas involving myofibers such as insulin resistance and myotoxicity of statins are mostly unresolved. The knowledge gained from this study may be used in the future to solve clinical problems related to the cell biology of the myofibers. / Tiivistelmä Luurankolihassolut eli myofiiberit ovat jättimäisiä monitumaisia soluja, jotka vastaavat lihassupistuksen aikaansaamisesta ja siten mahdollistavat jokapäiväisen liikkumisemme. Näiden suurten solujen rakenne poikkeaa selkeästi yksitumaisten solujen rakenteesta: myofiiberien tunnusomaisia piirteitä ovat kymmenet solun reunoille sijoittuneet tumat, tiiviisti pakkautunut supistumiskoneisto ja monimutkaisesti järjestynyt solukalvostojärjestelmä. Vaikka myofiiberien perusfysiologia tunnetaankin hyvin, niin tiedetään itse myofiiberien kalvostobiologiasta sangen vähän. Kokonaisuutena tämän tutkimuksen tarkoituksena oli tarkastella mRNA:n ja proteiinisynteesin sijaintia myofiibereissä. Lisäksi selvitimme lihassolujen kalvostodynamiikkaa. Tässä tutkimuksessa käytimme rotan flexor digitorum brevis (FDB) -lihaksesta saatua primääristä soluviljelymallia. Lisäksi hyödynsimme rotan extensor digitorum longus -lihaksesta hankittuja jääleikkeitä. Joissakin kokeissa käytimme myös myofiiberien esiastesoluja (myoblasteja ja myotuubeja). Immunohistokemian ja molekyylibiologian menetelmiä sovellettiin tutkimuksessa laajasti. Havaitsimme, että FDB –myofiibereissä mRNA sijaitsee aivan solukalvon alla. Proteiinisynteesi vaikutti olevan keskittynyt solukalvon alla sijaitsevien tumien ympärille, mutta myös solusisäisiin pistemäisiin rakenteisiin. Proteiinituotteet ylsivät satojen mikrometrien päähän alkuperäisestä tumastaan. Lisäksi proteiineille ei ilmennyt leviämisestettä myofiiberin sisäosiin. Leviämisen havaittiin olevan nopeaa sekä solulimassa että solulimakalvostoissa. Tutkiessamme solun eritystoimintaa huomasimme, että kuljetus ER:stä Golgin laitteeseen eroaa huomattavasti yksitumaisten solujen vastaavasta kuljetuksesta. Lopuksi havaitsimme myofiiberien pystyvän muodostamaan rasvapisaroita rasitusolosuhteissa. Rasvapisaroiden käyttäytyminen näytti myös poikkeavan siitä, mitä muissa soluissa on havaittu. Nykyään lihastutkimusta primäärisoluilla ei juuri tehdä maailmalla, minkä vuoksi myofiibereihin liittyvät lääketieteelliset pulmat kuten insuliiniresistenssi ja statiinien lihashaitat ovat suurelta osin ratkaisematta. Tästä tutkimuksesta saatuja tuloksia voitaneen jatkossa käyttää myofiiberien solubiologiaan liittyvien kliinisten ongelmien selvittämiseen.

Type II secretion system (T2SS) is one of the secretion systems found in gram-negative bacteria that provides transport of some bacterial proteins across the outer membrane. The passage through the membrane is mediated by a pore assembled from subunits called GspD or secretin. Together with three other components of T2SS, GspD was discovered in the genome of several protists including Naegleria gruberi, Andalucia godoyi, Reclinomonas americana, Neovahlkampfia damariscottae or in s species of genus Malawimonas. Previously it was found out that these proteins localize into the mitochondria. If found functional and with analogous topology to the bacterial system, the eukaryotic T2SS would represent unique mitochondrial protein export system. Secretin is essential subunit of T2SS which is not only the passive membrane channel, but also participates in the recognition of the substrate. Therefore, the research of the eukaryotic secretin could bring a valuable knowledge about the function of the mitochondrial T2SS. The experimental part of this thesis tries to characterize the eukaryotic secretin and it focuses on (i) the assembly of the secretin channel, in both, the bacteria and in the artificial membranes, (ii) the interactions of GspD with the other subunits of T2SS and (iii) the mechanism of import...

KCNE peptides are a class of type I transmembrane ß-subunits that assemble with and modulate the gating and ion conducting properties of a variety of voltage-gated K+ channels. Accordingly, mutations that affect the assembly and trafficking of K+ channel/KCNE complexes give rise to disease. The cellular mechanisms that oversee KCNE peptide assembly with voltage-gated K+ channels have yet to be elucidated. In Chapter II, we show that KCNE1 peptides are retained in the early stages of the secretory pathway until they co-assemble with KCNQ1 K+ channel subunits. Co-assembly with KCNQ1 channel subunits mediates efficient forward trafficking of KCNE1 peptides through the biosynthetic pathway and results in cell surface expression. KCNE1 peptides possess two N-linked glycosylation sites on their extracellular N-termini. Progression of KCNE1 peptides through the secretory pathway can be visualized through maturation of N-glycans attached to KCNE1. In Chapter III, we examine the kinetics and efficiency of N-linked glycan addition to KCNE1 peptides. Mutations that prevent glycosylation of KCNE1 give rise to the disorders of arrhythmia and deafness. We show that KCNE1 acquires N-glycans co- and post-translationally. Mutations that prevent N-glycosylation at the co-translational site have a long range effect on the disruption of post-translational glycosylation and suggest a novel biogenic mechanism for disease. In Chapter IV, we determine the presence of an additional post-translational modification on KCNE1 peptides. We define specific residues as sites of attachment of this modification identified as sialylated O-glycans and show that it occurs in native cardiac tissues where KCNE1 plays a role in the maintenance of cardiac rhythm. Taken together, these observations demonstrate the importance of having correctly assembled K+ channel/KCNE complexes at the cell surface for their proper physiological function and define a role for the posttranslational modifications of KCNE peptides in the proper assembly and trafficking of K+ channel/KCNE complexes.

The synapse, the minimal element required for interneuronal communication in the nervous sytems, is a structure with a great deal of plasticity, capable of undergoing changes that alter transmission strength, and even forming new connections. This property has great implications for a number of processes, including circuit formation and learning and memory. However, the proteins behind this synaptic plasticity are still not fully understood. To uncover and characterize the proteins that regulate the plastic nature of the synapse, I turned to the Drosophilalarval neuromuscular junction (NMJ), a powerful and accessible model system. I began by examining synaptic cell adhesion, as Cell Adhesion Molecules (CAMs) have long been implicated in synaptic outgrowth as well as learning and memory. CAMs have traditionally been thought of as molecules that mediate cell adhesion between the pre- and postsynaptic membrane. However, through the course of the studies presented here I demonstrate a CAM function that goes beyond simple cell adhesion, acting as a receptor that transduces adhesive signals to the intracellular space. In particular, I have demonstrated a role for the Drosophila CAM, Fasciclin II(FasII), in a signaling complex involving the Amyloid Precursor Protein-Like (APPL) and the Drosophila homolog of X11/MINT/Lin-10 (dX11). Further results show that deletion of either APPL or dX11 inhibits the FasII mediated outgrowth. These studies show that during NMJ expansion the transinteraction between FasII molecules in the pre- and postsynaptic membrane results in the recruitment of APPL and dX11 to the presynaptic cell surface, and the initiation of a signaling cascade that leads to bouton outgrowth. The next question addressed here was regarding the cytoskeletal changes that must occur during synapse remodeling. In particular I centered on the evolutionarily conserved cell polarity complex aPKC-Par3-Par6, which is know to regulate axon growth, the cell cytoskeleton during polarized cell division, and learning and memory. To understand the role of the cytoskeleton during NMJ expansion, I examined the organization of microtubules and actin during this process. Further, I identified atypical protein kinase C (aPKC) as a regulator of microtubule dynamics. I found that aPKC is required for regulating the degree of stabilization of synaptic microtubules. This stabilization requires the Microtubule Associated Protein-1B (MAP1B) homolog Futsch, which I demonstrated was required for aPKC to associate with and stabilize the microtubule cytoskeleton. The process of synaptic expansion not only requires modifications to the presynapse, but to the postsynapse as well. Previous work demonstrates that levels of the scaffolding proteins DrosophilaMembrane Associated Guanlyate Kinase (MAGUK) protein Discs-large (DLG), as well as the vertebrate homolog Postsynaptic Density-95 (PSD-95), which are concentrated at synapses, determine the size of postsynaptic membranes. To identify the underlying mechanisms of the regulation of postsynaptic size, we performed a yeast two hybrid screen, searching for DLG interacting proteins. We found a novel interaction between DLG, and a t-SNARE, GUK-interacting Syntaxin (Gtaxin; GTX), and went on to demonstrate that this interaction is required for proper postsynaptic membrane addition. Strong hypomorphic mutations in either dlg or gtx show a dramatic reduction in postsynaptic expansion. Overexpression of DLG produces an increase of synaptic GTX, as well as an increase in postsynaptic size, and an increased formation of GTX positive SNARE complexes. Taken together, these observations suggest that the MAGUK DLG regulates postsynaptic membrane addition by modulating the formation of a SNARE complex of the t-SNARE Gtaxin, and by targeting GTX to sites of postsynaptic membrane addition. In summary, the studies performed in this thesis probe a trans-synaptic adhesion based signaling complex required for presynaptic expansion, a specific pathway for dynamic microtubule stabilization required for pre- and postsynaptic expansion, and how a scaffolding protein regulates postsynaptic membrane expansion. These processes are all interconnected to maintain the efficacy of the synapse. The studies conducted revealed important information about how these processes are accomplished, and constitute an important step to elucidate the mechanisms by which synapse plasticity occurs at the level of single synaptic terminals.

The function of homologous DNA recombination in human mitochondria has been a topic of ongoing debate for many years, with implications for fields ranging from DNA repair and mitochondrial disease to population genetics. While genetic and biochemical evidence supports the presence of a mitochondrial recombination activity, the purpose for this activity and the proteins involved have remained elusive. The work presented in this thesis was designed to evaluate the mitochondrial localization of the major recombinase protein in human cells, Rad51, as well as determine what function it plays in the maintenance of mitochondrial DNA (mtDNA) copy number that is critical for production of chemical energy through aerobic respiration. The combination of subcellular fractionation with immunoblotting and immunoprecipitation approaches used in this study clearly demonstrates that Rad51 is a bona fide mitochondrial protein that localizes to the matrix compartment following oxidative stress, where it physically interacts with mtDNA. Rad51 was found to be critical for mtDNA copy number maintenance under stress conditions. This requirement for Rad51 was found to be completely dependent on ongoing mtDNA replication, as treatment with the DNA polymerase gamma (Pol ϒ) inhibitor, ddC, suppresses both recruitment of Rad51 to the mitochondria following the addition of stress, as well as the mtDNA degradation observed when Rad51 has been depleted from the cell. The data presented here support a model in which oxidative stress induces a three-part response: (1) The recruitment of repair factors including Rad51 to the mitochondrial matrix, (2) the activation of mtDNA degradation systems to eliminate extensively or persistently damaged mtDNA, and (3) the increase in mtDNA replication in order to maintain copy number. The stress-induced decrease in mtDNA copy number observed when Rad51 is depleted is likely the result of failure to stabilize or repair replication forks that encounter blocking lesions resulting in further damaged to the mtDNA and its eventual degradation.