Mitochondrial DNA Polymerase Research Articles

Human DNA polymerase η accommodates RNA for strand extension

Ribonucleotides are the natural analogs of deoxyribonucleotides, which can be misinserted by DNA polymerases, leading to the most abundant DNA lesions in genomes. During replication, DNA polymerases tolerate patches of ribonucleotides on the parental strands to different extents. The majority of human DNA polymerases have been reported to misinsert ribonucleotides into genomes. However, only PrimPol, DNA polymerase α, telomerase, and the mitochondrial human DNA polymerase (hpol) γ have been shown to tolerate an entire RNA strand. Y-family hpol η is known for translesion synthesis opposite the UV-induced DNA lesion cyclobutane pyrimidine dimer and was recently found to incorporate ribonucleotides into DNA. Here, we report that hpol η is able to bind DNA/DNA, RNA/DNA, and DNA/RNA duplexes with similar affinities. In addition, hpol η, as well as another Y-family DNA polymerase, hpol κ, accommodates RNA as one of the two strands during primer extension, mainly by inserting dNMPs opposite unmodified templates or DNA lesions, such as 8-oxo-2'-deoxyguanosine or cyclobutane pyrimidine dimer, even in the presence of an equal amount of the DNA/DNA substrate. The discovery of this RNA-accommodating ability of hpol η redefines the traditional concept of human DNA polymerases and indicates potential new functions of hpol η in vivo.

Iterated function systems for DNA replication.

The kinetic equations of DNA replication are shown to be exactly solved in terms of iterated function systems, running along the template sequence and giving the statistical properties of the copy sequences, as well as the kinetic and thermodynamic properties of the replication process. With this method, different effects due to sequence heterogeneity can be studied, in particular, a transition between linear and sublinear growths in time of the copies, and a transition between continuous and fractal distributions of the local velocities of the DNA polymerase along the template. The method is applied to the human mitochondrial DNA polymerase γ without and with exonuclease proofreading.

Plant organellar DNA primase-helicase synthesizes RNA primers for organellar DNA polymerases using a unique recognition sequence.

DNA primases recognize single-stranded DNA (ssDNA) sequences to synthesize RNA primers during lagging-strand replication. Arabidopsis thaliana encodes an ortholog of the DNA primase-helicase from bacteriophage T7, dubbed AtTwinkle, that localizes in chloroplasts and mitochondria. Herein, we report that AtTwinkle synthesizes RNA primers from a 5′-(G/C)GGA-3′ template sequence. Within this sequence, the underlined nucleotides are cryptic, meaning that they are essential for template recognition but are not instructional during RNA synthesis. Thus, in contrast to all primases characterized to date, the sequence recognized by AtTwinkle requires two nucleotides (5′-GA-3′) as a cryptic element. The divergent zinc finger binding domain (ZBD) of the primase module of AtTwinkle may be responsible for template sequence recognition. During oligoribonucleotide synthesis, AtTwinkle shows a strong preference for rCTP as its initial ribonucleotide and a moderate preference for rGMP or rCMP incorporation during elongation. RNA products synthetized by AtTwinkle are efficiently used as primers for plant organellar DNA polymerases. In sum, our data strongly suggest that AtTwinkle primes organellar DNA polymerases during lagging strand synthesis in plant mitochondria and chloroplast following a primase-mediated mechanism. This mechanism contrasts to lagging-strand DNA replication in metazoan mitochondria, in which transcripts synthesized by mitochondrial RNA polymerase prime mitochondrial DNA polymerase γ.

Abstract 1494: Interethnic germline variants in mitochondrial DNA polymerase (POLG1) induce mitochondrial dysfunction and confer tumorigenic properties

Abstract Purpose: Germline variants contribute to large fraction of the diversity observed among different ethnic groups and could give better insights in genetic predisposition and susceptibility from one population to another for certain disease including cancer. Decreased mitochondrial OXPHOS is one of the most common phenotypes of cancer cells. Mitochondrial DNA (mtDNA) depletion impairs OXPHOS and adaptive metabolic responses. In this context, DNA polymerase γ (POLG1) is known to function in human mitochondria and performs critical function of mtDNA replication and repair. POLG1 is the most frequent target of gene mutation and is involved in variety of mitochondrial diseases. POLG1 protein consists of exonuclease, linker and polymerase domain, each with distinct functions. Mutations in exonuclease domain leads to erroneous proofreading and is responsible for increased mutations in mtDNA whereas mutations in polymerase domain leads to erroneous replication and hence depletion in mtDNA. Germline variants in these domains of POLG1 may serve as important signature markers in association studies of individuals in a population study. Through our present study, we identified germline variants, examined copy number variation, expression and regulation of POLG1 gene in human cancers. We provide evidence that altered POLG1 expression as well as germline variations in POLG1 gene contribute to tumorigenesis. Methods: A comprehensive race based bioinformatics analysis of POLG1 gene in European-American and African-American was conducted and several unique germline mutations were discovered. To analyze functional contribution of these unique germline mutations in tumor progression, these unique prevalent mutations in the evolutionary conserved regions of exonuclease and polymerase domains we generated by site directed mutagenesis and functional and tumorigenic analysis was performed. Results: We observed marked differences in copy number variation of POLG1 through cBioPortal and Cosmic databases and validated the analysis in primary tumors and cancer cell lines. Our results also indicate that mtDNA copy number in cancer cell is governed by regulation of POLG1 methylation and demethylation status, suggestive of epigenetic regulation of POLG1. We identified a mitochondrial disease causing missense variation in polymerase domain of POLG1 at amino acid 1143 (E1143G) to be 25 times more prevalent in European-Americans when compared to African-Americans population. Expression of this germline variant (E1143G) increased glucose consumption, decreased ATP production and increased matrigel invasion. Discussion/ Conclusion: We demonstrate that POLG1 is epigenetically regulated and its unique germline variants contribute to disruption of mitochondrial function. Thus contributes to differences in increased predisposition to cancer in inter-ethnic population. Note: This abstract was not presented at the meeting. Citation Format: Prachi Bajpai, Bhupendra Singh, Kjerstin M. Owens, Vinodh Srinivasasainagendra, Hemant K. Tiwari, Keshav K. Singh. Interethnic germline variants in mitochondrial DNA polymerase (POLG1) induce mitochondrial dysfunction and confer tumorigenic properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1494. doi:10.1158/1538-7445.AM2017-1494

Speech and swallowing abnormalities in adults with POLG associated ataxia (POLG-A)

BackgroundMutations in the nuclear-encoded mitochondrial DNA polymerase gamma (POLG) can result in a wide spectrum of neurological deficits. A common presentation is progressive ataxia (POLG-A) which includes impaired speech and swallowing. The nature, severity and impact of these deficits in POLG-A is not known. A comprehensive quantitative and qualitative characterization of dysarthria and dysphagia in this recurrent ataxia disorder will assist in diagnostics, provide insights into the underlying pathology, and establish the foundation for future therapy trials. Methods14 consecutive patients with POLG (9 females, mean age=50.1y, SD=11.2) and 34 healthy controls were enrolled. Comprehensive assessments of motor speech and swallowing function, acoustic analysis of speech, videofluoroscopy and measures of quality of life were conducted. ResultsThe speech profile of individuals with POLG-A was characterized by poor control of pitch and strain-strangled voice quality, reduced rate of speech and longer variable silences between words, and articulatory breakdown including imprecise consonants and vowel distortions. Swallowing deficits included slower initiation of the swallow reflex, poor control of bolus and late epiglottic closure. Speech and swallowing related quality of life was worse than healthy controls. ConclusionsThe dysarthria and dysphagia profiles in POLG-A are largely symptomatic of impaired timing, indicating a mainly spinocerebellar deficit. Dysarthria and dysphagia contribute to a significant impairment in functional quality of life, and progress distinctly from other POLG-A dysfunctions like ataxia or cognitive impairment. Our assessments establish meaningful patient focused outcome measures that will be suitable for use in natural history studies and clinical trials.

Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML

AbstractMitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2′3′-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2′3′-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML.

Polymorphisms in POLG were associated with the prognosis and mtDNA content in hepatocellular carcinoma patients

POLG is a gene that codes for the catalytic subunit of the mitochondrial DNA polymerase, which is involved in the replication of mitochondrial DNA. Genetic variants in mitochondrial DNA polymerase-γ (POLG) have been associated with several malignancies. However, as an important metabolic tissue, association between genetic polymorphisms of POLG and the prognosis and mitochondrial DNA (mtDNA) content in hepatocellular carcinoma (HCC) remains unknown. Here we investigated the association between in POLG with the prognosis and mitochondrial DNA (mtDNA) content in hepatocellular carcinoma (HCC). Three nucleotide polymorphisms (SNPs) of rs1061316, rs2247233 and rs758130 in POLG were examined in 416 patients from two cohorts undergoing transcatheter arterial chemoembolization treatment. Leukocyte mtDNA content from 216 patients in cohort 2 was measured using a real-time PCR-based method. The association of SNPs with prognosis and of mtDNA content of patients was analyzed. The rs758130 in POLG gene was significantly associated with the prognosis of patients in a dose-dependent manner. Moreover, GG genotype in rs1061316 showed significantly high mtDNA content, an indicator of better prognosis. Our study for the first time demonstrates that rs1061316 and rs758130 in POLG is associated with the prognosis and leukocyte mtDNA content in HCC patients.

Inheritance: Male mtDNA Just Can’t Catch a Break

Mitochondrial DNA (mtDNA) is actively eliminated from the developing sperm in Drosophila. New work shows that the mitochondrial DNA polymerase, which normally replicates mtDNA, plays a surprising role in mtDNA elimination.

Synergistic Effects of the in cis T251I and P587L Mitochondrial DNA Polymerase γ Disease Mutations

Human mitochondrial DNA (mtDNA) polymerase γ (Pol γ) is the only polymerase known to replicate the mitochondrial genome. The Pol γ holoenzyme consists of the p140 catalytic subunit (POLG) and the p55 homodimeric accessory subunit (POLG2), which enhances binding of Pol γ to DNA and promotes processivity of the holoenzyme. Mutations within POLG impede maintenance of mtDNA and cause mitochondrial diseases. Two common POLG mutations usually found in cis in patients primarily with progressive external ophthalmoplegia generate T251I and P587L amino acid substitutions. To determine whether T251I or P587L is the primary pathogenic allele or whether both substitutions are required to cause disease, we overproduced and purified WT, T251I, P587L, and T251I + P587L double variant forms of recombinant Pol γ. Biochemical characterization of these variants revealed impaired DNA binding affinity, reduced thermostability, diminished exonuclease activity, defective catalytic activity, and compromised DNA processivity, even in the presence of the p55 accessory subunit. However, physical association with p55 was unperturbed, suggesting intersubunit affinities similar to WT. Notably, although the single mutants were similarly impaired, a dramatic synergistic effect was found for the double mutant across all parameters. In conclusion, our analyses suggest that individually both T251I and P587L substitutions functionally impair Pol γ, with greater pathogenicity predicted for the single P587L variant. Combining T251I and P587L induces extreme thermal lability and leads to synergistic nucleotide and DNA binding defects, which severely impair catalytic activity and correlate with presentation of disease in patients.

Enrichment of deleterious variants of mitochondrial DNA polymerase gene (POLG1) in bipolar disorder.

Rare missense variants, which likely account for a substantial portion of the genetic 'dark matter' for a common complex disease, are challenging because the impacts of variants on disease development are difficult to substantiate. This study aimed to examine the impacts of amino acid substitution variants in the POLG1 found in bipolar disorder, as an example and proof of concept, in three different modalities of assessment: in silico predictions, in vitro biochemical assays, and clinical evaluation. We then tested whether deleterious variants in POLG1 contributed to the genetics of bipolar disorder. We searched for variants in the POLG1 gene in 796 Japanese patients with bipolar disorder and 767 controls and comprehensively investigated all 23 identified variants in the three modalities of assessment. POLG1 encodes mitochondrial DNA polymerase and is one of the causative genes for a Mendelian-inheritance mitochondrial disease, which is occasionally accompanied by mood disorders. The healthy control data from the Tohoku Medical Megabank Organization were also employed. Although the frequency of carriers of deleterious variants varied from one method to another, every assessment achieved the same conclusion that deleterious POLG1 variants were significantly enriched in the variants identified in patients with bipolar disorder compared to those in controls. Together with mitochondrial dysfunction in bipolar disorder, the present results suggested deleterious POLG1 variants as a credible risk for the multifactorial disease.

The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T.

The mitochondrial toxicity of nucleoside reverse transcriptase inhibitors (NRTIs) is due to the inhibition of mitochondrial DNA (mtDNA) polymerase γ (pol γ). Previous studies have shown that wild type p53 (wtp53) can interact with pol γ and mtDNA to enhance mitochondrial DNA base excision repair (mtBER) activity and increase the accuracy of DNA synthesis. The N-terminal transactivation domain and central specific DNA-binding domain of p53 play critical roles in the stimulation of BER. In this study, we identified the possible roles of wtp53, Δ40p53 and Δ133p53 in regulating mtDNA pol γ activity in cells with d4T treatment. The results show that Δ40p53 and Δ133p53 can exist in mitochondrial fragments and form polymers with themselves or wtp53. Unlike wtP53, Δ133p53 alone cannot increase DNA pol γ activity. More importantly, we found that Δ133p53 played a negative role in p53 stimulation of DNA pol γ activity when studied in d4T-treated and d4T-untreated mitochondrial extracts. Gel shift data also indicate that Δ40p53 and Δ133p53 cannot interact with APE. Wtp53 and Δ40p53 can act antagonize the effect of d4T inhibition of DNA pol γ activity. However, when wtp53 interacted with Δ133p53, DNA pol γ activity was significantly decreased. Conclusion: Δ133p53 negatively regulates p53’s stimulation of pol γ in the presence and absence of d4T.

Differential Alterations of the Mitochondrial Morphology and Respiratory Chain Complexes during Postnatal Development of the Mouse Lung.

Mitochondrial biogenesis and adequate energy production in various organs of mammals are necessary for postnatal adaptation to extrauterine life in an environment with high oxygen content. Even though transgenic mice are frequently used as experimental models, to date, no combined detailed molecular and morphological analysis on the mitochondrial compartment in different lung cell types has been performed during postnatal mouse lung development. In our study, we revealed a significant upregulation of most mitochondrial respiratory complexes at protein and mRNA levels in the lungs of P15 and adult animals in comparison to newborns. The majority of adult animal samples showed the strongest increase, except for succinate dehydrogenase protein (SDHD). Likewise, an increase in mRNA expression for mtDNA transcription machinery genes (Polrmt, Tfam, Tfb1m, and Tfb2m), mitochondrially encoded RNA (mt-Rnr1 and mt-Rnr2), and the nuclear-encoded mitochondrial DNA polymerase (POLG) was observed. The biochemical and molecular results were corroborated by a parallel increase of mitochondrial number, size, cristae number, and complexity, exhibiting heterogeneous patterns in distinct bronchiolar and alveolar epithelial cells. Taken together, our results suggest a specific adaptation and differential maturation of the mitochondrial compartment according to the metabolic needs of individual cell types during postnatal development of the mouse lung.

Corrigendum: Platinated Nucleotides are Substrates for the Human Mitochondrial Deoxynucleotide Carrier (DNC) and DNA Polymerase γ: Relevance for the Development of New Platinum-Based Drugs.

Corrigendum: Platinated Nucleotides are Substrates for the Human Mitochondrial Deoxynucleotide Carrier (DNC) and DNA Polymerase γ: Relevance for the Development of New Platinum-Based Drugs.

Mitochondrial defects arise from nucleoside/nucleotide reverse transcriptase inhibitors in neurons: Potential contribution to HIV-associated neurocognitive disorders

The cornerstone of current HIV treatment is a class of drugs called nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs). However, patients who receive long term treatment with NRTIs often develop severe side effects, which are related to mitochondrial toxicity. The potential contribution of NRTI-mediated toxicity to HIV-associated neurocognitive disorders (HAND) has not been fully explored. NRTI toxicity is thought to be mediated through mitochondrial DNA polymerase γ (pol γ) inhibition, which impairs mitochondrial DNA (mtDNA) synthesis and leads to various mitochondrial dysfunctions. To evaluate the relationship between NRTI-mediated pol γ inhibition and mitochondrial toxicity in neurons, we systematically investigated mitochondrial regulation in NRTI-treated primary cortical neurons by measuring parameters related to mtDNA content, retrograde signaling responses and mitochondrial homeostasis. The effects of four different NRTIs with variable pol γ inhibitory activity and mitochondrial toxicity were assessed. The strong pol γ inhibitor, ddI, abolished mtDNA synthesis and greatly reduced mtDNA content. However, mtDNA transcription was not as severely affected, and no defects in oxidative phosphorylation were observed. Detrimental effects on mitochondrial respiration and motility were observed after AZT treatment in the absence of mtDNA depletion or inhibition of mtDNA synthesis. The results suggest that individual NRTIs, such as ddI and AZT, have the potential to cause mitochondrial toxicity in neurons. This mitochondrial toxicity would be expected to contribute to neurotoxicity in the central nervous system, and therefore, HAND etiology may be affected by NRTI treatment.

Characterizing the mitochondrial DNA polymerase gamma interactome by BioID identifies Ruvbl2 localizes to the mitochondria

Human mitochondrial DNA (mtDNA) is replicated by the mitochondrial DNA polymerase gamma (POLG). Using proximity dependent biotin labelling (BioID), we characterized the POLG interactome and identified new interaction partners involved in mtDNA maintenance, transcription, translation and protein quality control. We also identified interaction with the nuclear AAA+ ATPase Ruvbl2, suggesting mitochondrial localization for this protein. Ruvbl2 was detected in mitochondria-enriched fractions in leukemic cells. Additionally, transgenic overexpression of Ruvbl2 from an alternative translation initiation site resulted in mitochondrial co-localization. Overall, POLG interactome mapping identifies novel proteins which support mitochondrial biogenesis and a potential novel mitochondrial isoform of Ruvbl2.

Platinated Nucleotides are Substrates for the Human Mitochondrial Deoxynucleotide Carrier (DNC) and DNA Polymerase γ: Relevance for the Development of New Platinum‐Based Drugs.

Abstractcis‐[PtCl2(NH3)2] (cisplatin) is among the highest effective antitumor drugs used for the chemotherapeutic treatment of a broad range of malignancies. Recently, alongside with the classical direct bond to DNA, an alternative mechanism of action mediated by N7 platinated nucleotides has been suggested for cisplatin. Considering that mitochondria play an important role in cell death activation and in a significant portion of the clinical activity and pharmacological properties associated with cisplatin, aim of this research was to evaluate the possibility that platinated deoxynucleotides, as the model complex [Pt(dien)(N7‐5’‐dGTP)] (1), dien=diethylenetriamine, could be transported into mitochondria and then incorporated into mtDNA. The kinetic characterization has revealed that the mitochondrial deoxynucleotide carrier (DNC) transports complex 1 with high affinity. Finally, a highly efficient in organello DNA synthesis system, followed by ICP‐AES, has demonstrated that [Pt(dien)(N7‐5’‐dGTP)] is incorporated in the mitochondrial DNA by DNA polymerase γ. These results may have critical implications in the development of new generations of anticancer and/or antiviral nucleotide analogues with more specific cellular targets and fewer side effects.

Whole exome sequencing identifies a homozygous POLG2 missense variant in an infant with fulminant hepatic failure and mitochondrial DNA depletion

Mitochondrial DNA (mtDNA) depletion syndrome manifests as diverse early-onset diseases that affect skeletal muscle, brain and liver function. Mutations in several nuclear DNA-encoded genes cause mtDNA depletion. We report on a patient, a 3-month-old boy who presented with hepatic failure, and was found to have severe mtDNA depletion in liver and muscle. Whole-exome sequencing identified a homozygous missense variant (c.544C > T, p.R182W) in the accessory subunit of mitochondrial DNA polymerase gamma (POLG2), which is required for mitochondrial DNA replication. This variant is predicted to disrupt a critical region needed for homodimerization of the POLG2 protein and cause loss of processive DNA synthesis. Both parents were phenotypically normal and heterozygous for this variant. Heterozygous mutations in POLG2 were previously associated with progressive external ophthalmoplegia and mtDNA deletions. This is the first report of a patient with a homozygous mutation in POLG2 and with a clinical presentation of severe hepatic failure and mitochondrial depletion.

The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA

Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10-12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3'-TCC, TTC, and TTT, and the consensus sequence is 3'-Pu(Py)2-3 Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA.

Abstract 210: Targeting the mitochondrial DNA polymerase gamma with 2’3’-dideoxycytidine as a novel therapeutic strategy for acute myeloid leukemia

Abstract A subset of AML cells display unique mitochondrial characteristics such as increased mitochondrial DNA (mtDNA), mt mass, and sensitivity to inhibition of mtDNA transcription and protein translation. Given these unique mitochondrial characteristics, we evaluated the effect of inhibiting mtDNA replication by targeting the mtDNA polymerase gamma (POLG). POLG is a nuclear-encoded gene that replicates and repairs mtDNA. POLG mRNA is increased in leukemia cell lines and a subset of AML patients. We evaluated the preclinical efficacy of targeting POLG using the nucleoside analog 2’,3’-dideoxycytidine (ddC), an FDA-approved anti-retroviral drug that cross-reacts with POLG. Treatment with ddC at 500nM depleted mtDNA by >90%, decreased mtDNA-encoded COXI and COXII proteins and basal oxygen consumption, and induced apoptosis in AML cell lines (OCI-AML2, TEX, K562). ddC further decreased cell viability in a subset of primary AML cells, while normal peripheral blood stem cells (PBSC’s) were resistant to ddC in vitro. Next, we assessed the preclinical efficacy and toxicity of ddC in an OCI-AML2 xenograft model of human AML. Treatment with 35mg/kg of i.p. ddC induced tumor regression and decreased tumor mass by >75% compared to vehicle. No toxicity was observed including changes in liver enzymes or organ histology. We also observed reductions in COXI and COXII protein in tumors but not liver excised from treated mice. We next investigated the mechanism for preferential sensitivity to ddC in AML cells. A two-fold greater decrease in mtDNA content was observed in AML cells treated with ddC compared to PBSC’s, in part explaining the heightened sensitivity. However, the increased sensitivity to ddC in AML was not due to increased mtDNA turnover as mtDNA depletion after ethidium bromide treatment was comparable between AML and PBSC’s. Therefore, we investigated the role of ddC uptake and metabolism. ddC is transported by nucleoside transporters (ENT’s, CNT’s) and activated by cytoplasmic nucleoside kinases to ddC-triphosphate (ddC-TP). Analysis of Haferlach et. al. (2010) dataset demonstrated that a subset of AML cells have higher mRNA expression of hENT2 and hCNT1, and the nucleoside diphosphate kinases NME1,2,3,5 compared to normal hematopoietic cells. Using mass spectrometry, we observed higher levels of ddC and ddCTP in sensitive AML cells compared to resistant cells. Lastly, knockdown of the deoxycytidine kinase rescued ddC cytotoxicity in TEX cells. In summary, targeting POLG with ddC displays anti-leukemic activity in both in vitro and in vivo models of AML. The preferential toxicity of ddC is due, in part, to increased rates of uptake and activation in AML along as well as their increased sensitivity to inhibition of oxidative phosphorylation. Thus, POLG inhibitors such as ddC may have clinical utility in a subset of AML. Citation Format: Sanduni Liyanage, Rose Hurren, Xiaoming Wang, Neil Maclean, Rebecca Laposa, Aaron Schimmer. Targeting the mitochondrial DNA polymerase gamma with 2’3’-dideoxycytidine as a novel therapeutic strategy for acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 210.

Oxidative DNA damage stalls the human mitochondrial replisome.

Oxidative stress is capable of causing damage to various cellular constituents, including DNA. There is however limited knowledge on how oxidative stress influences mitochondrial DNA and its replication. Here, we have used purified mtDNA replication proteins, i.e. DNA polymerase γ holoenzyme, the mitochondrial single-stranded DNA binding protein mtSSB, the replicative helicase Twinkle and the proposed mitochondrial translesion synthesis polymerase PrimPol to study lesion bypass synthesis on oxidative damage-containing DNA templates. Our studies were carried out at dNTP levels representative of those prevailing either in cycling or in non-dividing cells. At dNTP concentrations that mimic those in cycling cells, the replication machinery showed substantial stalling at sites of damage, and these problems were further exacerbated at the lower dNTP concentrations present in resting cells. PrimPol, the translesion synthesis polymerase identified inside mammalian mitochondria, did not promote mtDNA replication fork bypass of the damage. This argues against a conventional role for PrimPol as a mitochondrial translesion synthesis DNA polymerase for oxidative DNA damage; however, we show that Twinkle, the mtDNA replicative helicase, is able to stimulate PrimPol DNA synthesis in vitro, suggestive of an as yet unidentified role of PrimPol in mtDNA metabolism.