Role of apoptosis-inducing factor (AIF) in programmed nuclear death during conjugation in Tetrahymena thermophila

<p>Abstract</p> <p>Background</p> <p>Programmed nuclear death (PND), which is also referred to as nuclear apoptosis, is a remarkable process that occurs in ciliates during sexual reproduction (conjugation). In <it>Tetrahymena thermophila</it>, when the new macronucleus differentiates, the parental macronucleus is selectively eliminated from the cytoplasm of the progeny, concomitant with apoptotic nuclear events. However, the molecular mechanisms underlying these events are not well understood. The parental macronucleus is engulfed by a large autophagosome, which contains numerous mitochondria that have lost their membrane potential. In animals, mitochondrial depolarization precedes apoptotic cell death, which involves DNA fragmentation and subsequent nuclear degradation.</p> <p>Results</p> <p>We focused on the role of mitochondrial apoptosis-inducing factor (AIF) during PND in <it>Tetrahymena</it>. The disruption of <it>AIF </it>delays the normal progression of PND, specifically, nuclear condensation and kilobase-size DNA fragmentation. AIF is localized in <it>Tetrahymena </it>mitochondria and is released into the macronucleus prior to nuclear condensation. In addition, AIF associates and co-operates with the mitochondrial DNase to facilitate the degradation of kilobase-size DNA, which is followed by oligonucleosome-size DNA laddering.</p> <p>Conclusions</p> <p>Our results suggest that <it>Tetrahymena </it>AIF plays an important role in the degradation of DNA at an early stage of PND, which supports the notion that the mitochondrion-initiated apoptotic DNA degradation pathway is widely conserved among eukaryotes.</p

Repair of Oxidative DNA Damage and Cancer: Recent Progress in DNA Base Excision Repair

SIGNIFICANCE: Reactive oxygen species (ROS) are generated by exogenous and environmental genotoxins, but also arise from mitochondria as byproducts of respiration in the body. ROS generate DNA damage of which pathological consequence, including cancer is well established. Research efforts are intense to understand the mechanism of DNA base excision repair, the primary mechanism to protect cells from genotoxicity caused by ROS. RECENT ADVANCES: In addition to the notion that oxidative DNA damage causes transformation of cells, recent studies have revealed how the mitochondrial deficiencies and ROS generation alter cell growth during the cancer transformation. CRITICAL ISSUES: The emphasis of this review is to highlight the importance of the cellular response to oxidative DNA damage during carcinogenesis. Oxidative DNA damage, including 7,8-dihydro-8-oxoguanine, play an important role during the cellular transformation. It is also becoming apparent that the unusual activity and subcellular distribution of apurinic/apyrimidinic endonuclease 1, an essential DNA repair factor/redox sensor, affect cancer malignancy by increasing cellular resistance to oxidative stress and by positively influencing cell proliferation. FUTURE DIRECTIONS: Technological advancement in cancer cell biology and genetics has enabled us to monitor the detailed DNA repair activities in the microenvironment. Precise understanding of the intracellular activities of DNA repair proteins for oxidative DNA damage should provide help in understanding how mitochondria, ROS, DNA damage, and repair influence cancer transformation

Base excision repair and the role of MUTYH

The correction of exogenous and endogenous environmental insult to DNA involves a series of DNA repair mechanisms that reduce the likelihood of mutation accumulation and hence an increased probability of tumour development. The mechanisms underlying the process of base excision repair are relatively well understood and are placed in context with how deterioration of this process is associated with an increased risk of malignancy

Analysis of nuclear transport signals in the human apurinic/apyrimidinic endonuclease (APE1/Ref1)

The mammalian abasic-endonuclease1/redox-factor1 (APE1/Ref1) is an essential protein whose subcellular distribution depends on the cellular physiological status. However, its nuclear localization signals have not been studied in detail. We examined nuclear translocation of APE1, by monitoring enhanced green fluorescent protein (EGFP) fused to APE1. APE1's nuclear localization was significantly decreased by deleting 20 amino acid residues from its N-terminus. Fusion of APE1's N-terminal 20 residues directed nuclear localization of EGFP. An APE1 mutant lacking the seven N-terminal residues (ND7 APE1) showed nearly normal nuclear localization, which was drastically reduced when the deletion was combined with the E12A/D13A double mutation. On the other hand, nearly normal nuclear localization of the full-length E12A/D13A mutant suggests that the first 7 residues and residues 8–13 can independently promote nuclear import. Both far-western analyses and immuno-pull-down assays indicate interaction of APE1 with karyopherin alpha 1 and 2, which requires the 20 N-terminal residues and implicates nuclear importins in APE1's nuclear translocation. Nuclear accumulation of the ND7 APE1(E12A/D13A) mutant after treatment with the nuclear export inhibitor leptomycin B suggests the presence of a previously unidentified nuclear export signal, and the subcellular distribution of APE1 may be regulated by both nuclear import and export

Yeast apurinic/apyrimidinic endonuclease Apn1 protects mammalian neuronal cell line from oxidative stress

Reactive oxygen species (ROS) have been implicated as one of the agents responsible for many neurodegenerative diseases. A critical target for ROS is DNA. Most oxidative stress-induced DNA damage in the nucleus and mitochondria is removed by the base excision repair pathway. Apn1 is a yeast enzyme in this pathway which possesses a wider substrate specificity and greater enzyme activity than its mammalian counterpart for removing DNA damage, making it a good therapeutic candidate. For this study we targeted Apn1 to mitochondria in a neuronal cell line derived from the substantia nigra by using a mitochondrial targeting signal (MTS) in an effort to hasten the removal of DNA damage and thereby protect these cells. We found that following oxidative stress, mitochondrial DNA (mtDNA) was repaired more efficiently in cells containing Apn1 with the MTS than controls. There was no difference in nuclear repair. However, cells that expressed Apn1 without the MTS showed enhanced repair of both nuclear and mtDNA. Both Apn1-expressing cells were more resistant to cell death following oxidative stress compared with controls. Therefore, these results reveal that the expression of Apn1 in neurons may be of potential therapeutic benefit for treating patients with specific neurodegenerative diseases

Plant mitochondria possess a short-patch base excision DNA repair pathway

Despite constant threat of oxidative damage, sequence drift in mitochondrial and chloroplast DNA usually remains very low in plant species, indicating efficient defense and repair. Whereas the antioxidative defense in the different subcellular compartments is known, the information on DNA repair in plant organelles is still scarce. Focusing on the occurrence of uracil in the DNA, the present work demonstrates that plant mitochondria possess a base excision repair (BER) pathway. In vitro and in organello incision assays of double-stranded oligodeoxyribonucleotides showed that mitochondria isolated from plant cells contain DNA glycosylase activity specific for uracil cleavage. A major proportion of the uracil–DNA glycosylase (UDG) was associated with the membranes, in agreement with the current hypothesis that the DNA is replicated, proofread and repaired in inner membrane-bound nucleoids. Full repair, from uracil excision to thymidine insertion and religation, was obtained in organello following import of a uracil-containing DNA fragment into isolated plant mitochondria. Repair occurred through single nucleotide insertion, which points to short-patch BER. In vivo targeting and in vitro import of GFP fusions showed that the putative UDG encoded by the At3g18 630 locus might be the first enzyme of this mitochondrial pathway in Arabidopsis thaliana

The Functions of Human Dna2 in Mitochondrial and Nuclear DNA Maintenance

Coordination between DNA replication, DNA repair and cell-cycle progression ensures high fidelity DNA replication thus preventing mutations and DNA rearrangements. Interestingly, in addition to nuclear DNA stability, mitochondrial DNA: mtDNA) integrity is also essential for normal development. The current challenge resides in unraveling the different mechanisms that govern nuclear and mtDNA stability and to understand how these two separated genomes have evolved. This work focuses on delineating the biological functions of human Dna2: hDna2). Dna2 is a highly conserved helicase/nuclease that in yeast participates in DNA replication and Okazaki fragment maturation, DNA repair, and telomere maintenance. Immunofluorescence and biochemical fractionation studies demonstrated that in addition to its nuclear localization, hDna2 is also present inside the mitochondria where it colocalized with a subfraction of DNA-containing mitochondrial nucleoids in unperturbed cells. Upon the expression of disease-associated mutant forms of the mitochondrial Twinkle helicase, which induce DNA replication pausing/stalling, hDna2 accumulated within nucleoids suggesting that it participates in mtDNA replication/repair. In accordance with these observations, RNA interference-mediated depletion of hDna2 led to a decrease in mtDNA replication intermediates and inefficient repair of damaged mtDNA. I have also investigated the nuclear function of hDna2 and demonstrate that it participates in DNA replication. RNAi- mediated depletion of hDna2 led to nuclear genomic instability that is accompanied by the activation of the replication checkpoint kinase Chk1 in late S/G2 phase. Genetic rescue experiments revealed that both hDna2\u27s nuclease and helicase activities are essential to maintain genomic stability, and suggest that these activities are coupled on long DNA flaps that arise during Okazaki fragments maturation. Furthermore, observations that hDna2 interacts with a member of the replisome, And-1, in a replication dependent manner, suggests that hDna2 is recruited to replication sites and actively participates in DNA replication. In accordance with biochemical and genetic models that predict that Dna2\u27s activity is only required for a small percent of flaps that escape the activities of FEN1, hDna2 depletion did not result in slower maturation of newly synthesized DNA. In contrast, FEN1-depleted cells did result in slower maturation confirming that FEN1 is the main flap endonuclease that processes Okazaki fragments into ligable nicks. To establish whether hDna2 participates in DNA replication fork progression, we analyzed track length of replicating forks in vivo using micro-fluidic- assisted replication track analysis: maRTA). Surprisingly, we did not observe slowing of the replication fork upon hDna2 or FEN1 depletion suggesting that replication fork progression is insensitive to Okazaki fragment maturation. However, maRTA analysis revealed that origin firing events are reduced upon hDna2 depletion suggesting that hDna2 also participates in the firing of replication origins. In agreement with this hypothesis, chromatin immunoprecipitation: ChIP) analysis revealed that hDna2 specifically localizes to replication origins. Altogether, the work presented here demonstrates that hDna2 is a novel addition to the growing list of proteins that participate in both nuclear and mtDNA maintenance and further suggests that mechanisms of DNA replication/repair are conserved between both organelles. Furthermore, this work increases our understanding of the molecular mechanisms that ensure high fidelity replication and provides novel avenues in our quest to understand human diseases caused by mutations in DNA replication genes

The mitochondrial nature of the DNA repair protein APE1

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. Apart from its canonical activities, recent studies have demonstrated that APE1 is also enzymatically active on RNA molecules. The present study unveils for the first time a new role of APE1 in the metabolism of RNA in mitochondria. Our data demonstrate that APE1 binds and exerts endoribonuclease activity on abasic mitochondrial messenger RNA. Loss of APE1 determines the accumulation of damaged mitochondrial mRNA species determining impairment in protein translation and reduced expression of mitochondrial encoded proteins, finally leading to less efficient mitochondrial respiration. All these effects are rescued by the expression of a recombinant mitochondrial targeted form of APE1 protein. Altogether, our data demonstrate that APE1 has an active role in the degradation of the mitochondrial mRNAs and a profound impact on mitochondria well-being