Nuclear Genome Instability Research Articles

Yeast EndoG prevents genome instability by degrading extranuclear DNA species

In metazoans mitochondrial DNA (mtDNA) or retrotransposon cDNA released to cytoplasm are degraded by nucleases to prevent sterile inflammation. It remains unknown whether degradation of these DNA also prevents nuclear genome instability. We used an amplicon sequencing-based method in yeast enabling analysis of millions of DSB repair products. In non-dividing stationary phase cells, Pol4-mediated non-homologous end-joining increases, resulting in frequent insertions of 1–3 nucleotides, and insertions of mtDNA (NUMTs) or retrotransposon cDNA. Yeast EndoG (Nuc1) nuclease limits insertion of cDNA and transfer of very long mtDNA ( >10 kb) to the nucleus, where it forms unstable circles, while promoting the formation of short NUMTs (~45–200 bp). Nuc1 also regulates transfer of extranuclear DNA to nucleus in aging or meiosis. We propose that Nuc1 preserves genome stability by degrading retrotransposon cDNA and long mtDNA, while short NUMTs originate from incompletely degraded mtDNA. This work suggests that nucleases eliminating extranuclear DNA preserve genome stability.

The increase in cell death rates in caloric restricted cells of the yeast helicase mutant rrm3 is Sir complex dependent

Calorie restriction (CR), which is a reduction in calorie intake without malnutrition, usually extends lifespan and improves tissue integrity. This report focuses on the relationship between nuclear genomic instability and dietary-restriction and its effect on cell survival. We demonstrate that the cell survival rates of the genomic instability yeast mutant rrm3 change under metabolic restricted conditions. Rrm3 is a DNA helicase, chromosomal replication slows (and potentially stalls) in its absence with increased rates at over 1400 natural pause sites including sites within ribosomal DNA and tRNA genes. Whereas rrm3 mutant cells have lower cell death rates compared to wild type (WT) in growth medium containing normal glucose levels (i.e., 2%), under CR growth conditions cell death rates increase in the rrm3 mutant to levels, which are higher than WT. The silent-information-regulatory (Sir) protein complex and mitochondrial oxidative stress are required for the increase in cell death rates in the rrm3 mutant when cells are transferred from growth medium containing 2% glucose to CR-medium. The Rad53 checkpoint protein is highly phosphorylated in the rrm3 mutant in response to genomic instability in growth medium containing 2% glucose. Under CR, Rad53 phosphorylation is largely reduced in the rrm3 mutant in a Sir-complex dependent manner. Since CR is an adjuvant treatment during chemotherapy, which may target genomic instability in cancer cells, our studies may gain further insight into how these therapy strategies can be improved.

Mitochondrial superoxide dismutase Sod2 suppresses nuclear genome instability during oxidative stress.

Oxidative stress can damage DNA and thereby contribute to genome instability. To avoid an imbalance or overaccumulation of reactive oxygen species (ROS), cells are equipped with antioxidant enzymes that scavenge excess ROS. Cells lacking the RecQ-family DNA helicase Sgs1, which contributes to homology-dependent DNA break repair and chromosome stability, are known to accumulate ROS, but the origin and consequences of this oxidative stress phenotype are not fully understood. Here, we show that the sgs1 mutant exhibits elevated mitochondrial superoxide, increased mitochondrial mass, and accumulation of recombinogenic DNA lesions that can be suppressed by antioxidants. Increased mitochondrial mass in the sgs1Δ mutant is accompanied by increased mitochondrial branching, which was also inducible in wildtype cells by replication stress. Superoxide dismutase Sod2 genetically interacts with Sgs1 in the suppression of nuclear chromosomal rearrangements under paraquat (PQ)-induced oxidative stress. PQ-induced chromosome rearrangements in the absence of Sod2 are promoted by Rad51 recombinase and the polymerase subunit Pol32. Finally, the dependence of chromosomal rearrangements on the Rev1/Pol ζ mutasome suggests that under oxidative stress successful DNA synthesis during DNA break repair depends on translesion DNA synthesis.

Downregulation of YAP Activity Restricts P53 Hyperactivation to Promote Cell Survival in Confinement.

Cell migration through confining three dimensional (3D) topographies can lead to loss of nuclear envelope integrity, DNA damage, and genomic instability. Despite these detrimental phenomena, cells transiently exposed to confinement do not usually die. Whether this is also true for cells subjected to long-term confinement remains unclear at present. To investigate this, photopatterning and microfluidics are employed to fabricate a high-throughput device that circumvents limitations of previous cell confinement models and enables prolonged culture of single cells in microchannels with physiologically relevant length scales. The results of this study show that continuous exposure to tight confinement can trigger frequent nuclear envelope rupture events, which in turn promote P53 activation and cell apoptosis. Migrating cells eventually adapt to confinement and evade cell death by downregulating YAP activity. Reduced YAP activity, which is the consequence of confinement-induced YAP1/2 translocation to the cytoplasm, suppresses the incidence of nuclear envelope rupture and abolishes P53-mediated cell death. Cumulatively, this work establishes advanced, high-throughput biomimetic models for better understanding cell behavior in health and disease, and underscores the critical role of topographical cues and mechanotransduction pathways in the regulation of cell life and death.

AKT2-mediated nuclear deformation leads to genome instability during epithelial-mesenchymal transition

Nuclear deformation has been observed in some cancer cells for decades, but its underlying mechanism and biological significance remain elusive. To address these questions, we employed human lung cancer A549 cell line as a model in context with transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition. Here, we report that nuclear deformation induced by TGFβ is concomitant with increased phosphorylation of lamin A at Ser390, defective nuclear lamina and genome instability. AKT2 and Smad3 serve as the downstream effectors for TGFβ to induce nuclear deformation. AKT2 directly phosphorylates lamin A at Ser390, whereas Smad3 is required for AKT2 activation upon TGFβ stimulation. Expression of the lamin A mutant with a substitution of Ser390 to Ala or suppression of AKT2 or Smad3 prevents nuclear deformation and genome instability induced by TGFβ. These findings reveal a molecular mechanism for TGFβ-induced nuclear deformation and establish a role of nuclear deformation in genome instability during epithelial-mesenchymal transition.

Aging-associated accumulation of mitochondrial DNA mutations in tumor origin

Abstract The majority of cancer patients are among aged population, suggesting an urgent need to advance our knowledge on complicated relationship between aging and cancer. It has been hypothesized that metabolic changes during aging could act as a driver for tumorigenesis. Given the fact that mitochondrial DNA (mtDNA) mutations are common in both tumors and aged tissues, it is interesting to contemplate possible role of age-related mtDNA mutations in tumorigenesis. MtDNA encodes genes essential for mitochondrial metabolism, and mtDNA mutates at a much higher rate than nuclear genome. Random drifting of somatic mtDNA mutations, as a result of cell division or mitochondrial turnover during aging, may lead to more and more cells harboring high-frequency pathogenic mtDNA mutations, albeit at different loci, in single-cells. Such mutations can induce metabolic reprogramming, nuclear genome instability and immune response, which might increase the likelihood of tumorigenesis. In this review, we summarize current understanding of how mtDNA mutations accumulate with aging and how these mutations could mechanistically contribute to tumor origin. We also discuss potential prevention strategies for mtDNA mutation-induced tumorigenesis, and future works needed in this direction.

Base excision repair causes age-dependent accumulation of single-stranded DNA breaks that contribute to Parkinson disease pathology.

SummaryAging, genomic stress, and mitochondrial dysfunction are risk factors for neurodegenerative pathologies, such as Parkinson disease (PD). Although genomic instability is associated with aging and mitochondrial impairment, the underlying mechanisms are poorly understood. Here, we show that base excision repair generates genomic stress, promoting age-related neurodegeneration in a Caenorhabditis elegans PD model. A physiological level of NTH-1 DNA glycosylase mediates mitochondrial and nuclear genomic instability, which promote degeneration of dopaminergic neurons in older nematodes. Conversely, NTH-1 deficiency protects against α-synuclein-induced neurotoxicity, maintaining neuronal function with age. This apparent paradox is caused by modulation of mitochondrial transcription in NTH-1-deficient cells, and this modulation activates LMD-3, JNK-1, and SKN-1 and induces mitohormesis. The dependance of neuroprotection on mitochondrial transcription highlights the integration of BER and transcription regulation during physiological aging. Finally, whole-exome sequencing of genomic DNA from patients with idiopathic PD suggests that base excision repair might modulate susceptibility to PD in humans.

Tyrosine phosphorylation of lamin A by Src promotes disassembly of nuclear lamina in interphase.

Lamins form the nuclear lamina, which is important for nuclear structure and activity. Although posttranslational modifications, in particular serine phosphorylation, have been shown to be important for structural properties and functions of lamins, little is known about the role of tyrosine phosphorylation in this regard. In this study, we found that the constitutively active Src Y527F mutant caused the disassembly of lamin A/C. We demonstrate that Src directly phosphorylates lamin A mainly at Tyr45 both in vitro and in intact cells. The phosphomimetic Y45D mutant was diffusively distributed in the nucleoplasm and failed to assemble into the nuclear lamina. Depletion of lamin A/C in HeLa cells induced nuclear dysmorphia and genomic instability as well as increased nuclear plasticity for cell migration, all of which were partially restored by re-expression of lamin A, but further promoted by the Y45D mutant. Together, our results reveal a novel mechanism for regulating the assembly of nuclear lamina through Src and suggest that aberrant phosphorylation of lamin A by Src may contribute to nuclear dysmorphia, genomic instability, and nuclear plasticity.

Nuclear and mitochondrial genome instability induced by fractions of ethanolic extract from Hovenia dulcis Thunberg in Saccharomyces cerevisiae strains.

Hovenia dulcis is a plant commonly used as a pharmaceutical supplement, having displayed important pharmacological properties such antigiardic, antineoplastic and hepatoprotective. The purpose of this work was investigate the cytotoxic, genotoxic and mutagenic potential from fractions of Hovenia dulcis ethanolic extract on Saccharomyces cerevisiae strains FF18733 (wild type) and CD138 (ogg1). Ethanolic extract from Hovenia dulcis leaves was fractioned using organic solvents according to increasing polarity: Hexane (1:1), dichlorometane (1:1), ethyl acetate (1:1) and butanol (1:1). Three experimental assays were performed, such as (i) inactivation of cultures; (ii) mutagenesis (canavanine resistance system) and (iii) loss of mitochondrial function (petites colonies). The findings shown a decrease in cell viability in FF18733 and CD138 strains; all fractions of the extract were mutagenic in CD138 strain; only ethyl acetate and butanol fractions increased the rate of petites colonies for CD138 strains. Ethyl acetate and n-butanol fractions induces mutagenicity, at the evaluated concentrations, in mitochondrial and genomic DNA in CD138 strain, mediated by oxidative lesions. In conclusion, it is possible to infer that the lesions caused by the extract fractions could be mediated by reactive oxygen species and might reach multiple molecular targets to cause cellular damage.

Dysregulation of hsa-miR-34a and hsa-miR-449a leads to overexpression of PACS-1 and loss of DNA damage response (DDR) in cervical cancer

We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3' terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response, S-phase cell cycle arrest, and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, Lys382-p53 acetylation, and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or -449a or through PACS-1 cDNA transfection led to the reversal of DNA damage response and restoration of cell growth. Release of cells post 24-h serum starvation showed PACS-1 nuclear localization at G1-S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response, resulting in the development of chemo-resistant tumors.

Abstract PO-076: miR-181a initiates and perpetuates ovarian cancer transformation through inhibition of the tumor suppressors RB1 and stimulator of interferon genes (STING)

Abstract Genomic instability predisposes cells to malignant transformation, however the molecular mechanisms that allow for the propagation of cells with a high-degree of genomic instability remains unclear. Here we report that miR-181a is able to transform fallopian tube secretory epithelial cells (FTSECs, the precursor cell type for the majority of high-grade serous ovarian cancers) through the inhibition of RB1. Increased miR-181a expression simultaneously drives cell protective inhibition of the stimulator-of-interferon-genes (STING) in order to maintain a microenvironment conducive to the propagation of cells with a high-degree of genomic instability. We found that miR-181a inhibition of RB1 leads to profound nuclear defects, genomic instability, and nuclear rupture resulting in a persistence of genomic material in the cytoplasm. Normally, this persistence of genomic material in the cytoplasm induces a cell-intrinsic interferon response through STING to drive cell death. However, miR-181a directly downregulates STING and prevents interferon induction and cell death in the FTSECs. The most common mechanism by which oncogenic miRNAs promote tumorigenesis is through the direct inhibition of tumor suppressor genes. However, our studies highlight a new mechanism of oncomiR transformation through the combination of tumor suppressor gene and intrinsic interferon signaling inhibition that initiates and propagates tumor cell survival. Importantly, we found that miR-181a induction in ovarian patient tumors is tightly associated with decreased IFNγ response and downregulation of lymphocyte infiltration and leukocyte fraction. Our findings are the first to identify a miRNA, miR-181a, that can downregulate STING expression and suppress activation of cell-intrinsic innate immune signaling in precursor cells undergoing oncogenic transformation to create a survival advantage. Our studies support the notion that the induction of STING-mediated signaling in developing cancer cells could lead directly to cancer cell death, and that these effects can be inhibited by miR-181a. Given the recent interest in the development of STING agonists as a strategy to harness the immune system to treat cancer, this study introduces miR-181a as a novel patient selective biomarker as well as potential therapeutic target for HGSOC treatment. Citation Format: Matthew Knarr, Rita Avelar, Sreeja Sekhar, Lily Kwiatkowski, Michele Dziubinski, Jessica McAnulty, Stephanie Skala, Stefanie Avril, Ronny Drapkin, Analisa DiFeo. miR-181a initiates and perpetuates ovarian cancer transformation through inhibition of the tumor suppressors RB1 and stimulator of interferon genes (STING) [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-076.

Abstract B02: miR-181a initiates and perpetuates oncogenic transformation through the regulation of innate immune signaling

Abstract Genomic instability predisposes cells to malignant transformation; however, the molecular mechanisms that allow for the propagation of cells with a high degree of genomic instability remain unclear. Here we report that miR-181a is able to transform fallopian tube secretory epithelial cells—the precursor cell type for the majority of high-grade serous ovarian cancers—through the inhibition of RB1 and simultaneously drives a cell-protective inhibition of the stimulator-of-interferon-genes (STING) in order to maintain a microenvironment conducive to the propagation of cells with a high-degree of genomic instability. We found that miR-181a inhibition of RB1 leads to profound nuclear defects, genomic instability, and nuclear rupture resulting in a persistence of genomic material in the cytoplasm. While normally, this persistence of genomic material in the cytoplasm induces interferon response through STING to drive cell death, miR-181a directly downregulates STING and prevents apoptosis. The most common mechanism by which oncogenic miRNAs promote tumorigenesis is through the direct inhibition of tumor suppressor genes; however, our studies highlight a new mechanism of oncomiR transformation through the combination of tumor suppressor gene inhibition and abrogation of immune surveillance that initiates and propagates tumor cell survival. Importantly, we found that miR-181a induction in human ovarian tumors is tightly associated with decreased IFNg response and downregulation of lymphocyte infiltration and leukocyte fraction. To date, DNA oncoviruses are the only known inhibitors of STING that allow for cellular transformation; thus, our findings are the first to identify a genetic factor, miR-181a, that can downregulate STING expression and impair signaling in cancer cells, creating a survival advantage. Our studies support the notion that the induction of STING-mediated signaling in cancer cells could lead directly to cancer cell death; however, these effects are abrogated by miR-181a. Given the recent interest in the development of STING agonists as a strategy to harness the immune system to treat cancer, this study introduces a novel patient-selective biomarker as well as potent therapeutic target for development of the most effective combination treatments. Citation Format: Matthew Knarr, Lily J. Kwiatkowski, Michele Dziubinski, Jessica McAnulty, Stephanie Skala, Stefanie Avril, Ronny Drapkin, Analisa DiFeo. miR-181a initiates and perpetuates oncogenic transformation through the regulation of innate immune signaling [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B02.

Trypanosoma brucei ATR Links DNA Damage Signaling during Antigenic Variation with Regulation of RNA Polymerase I-Transcribed Surface Antigens

SummaryTrypanosoma brucei evades mammalian immunity by using recombination to switch its surface-expressed variant surface glycoprotein (VSG), while ensuring that only one of many subtelomeric multigene VSG expression sites are transcribed at a time. DNA repair activities have been implicated in the catalysis of VSG switching by recombination, not transcriptional control. How VSG switching is signaled to guide the appropriate reaction or to integrate switching into parasite growth is unknown. Here, we show that the loss of ATR, a DNA damage-signaling protein kinase, is lethal, causing nuclear genome instability and increased VSG switching through VSG-localized damage. Furthermore, ATR loss leads to the increased transcription of silent VSG expression sites and expression of mixed VSGs on the cell surface, effects that are associated with the altered localization of RNA polymerase I and VEX1. This work shows that ATR acts in antigenic variation both through DNA damage signaling and surface antigen expression control.

ВТОРИННИЙ РІСТ У ДИХАЛЬНО-ДЕФІЦИТНОМУ ШТАМІ ДРІЖДЖІВ SACCHAROMYCES CEREVISIAE ВНАСЛІДОК ДЕЛЕЦІЇ ГЕНА YKU70

It is known that significant causes of malignant tumors are destabilization of the nuclear genome and mitochondrial dysfunction. Adaptive regrowth in yeast colonies (the appearance of cell subpopulations more adapted to unfavorable conditions under conditions of the death of the original culture) is used as a model of the initial stages of carcinogenesis. To study the features of the formation of adaptive regrowth, a reparationdefective and respiratory-deficient yeast strain of Saccharomyces cerevisiae was created. The thermosensitive mutation in the yku70 gene was used as an inducer of nuclear genome instability (at 37 оC it causes cell cycle arrest due to a reduction of the length of telomeric regions of chromosomes). Damage to the mitochondrial DNA of the ∆yku70 strain led to its respiratory deficiency (petite mutation). The isolated petite mutant ∆yku70 strain was cultured at optimal 28 оC and restrictive 37 оC temperatures, the state of the cell suspension was evaluated by light and fluorescence microscopy, to determine the viability of cells was used the analysis of microcolonies growth. Isolation of adaptive regrowth clones and analysis of their properties by the method of serial dilutions were conducted. To assess the genome stability of selected clones of adaptive regrowth, PCR analysis of the microsatellite sequences YOR267C, SC8132X, SCPTSY7 was conducted. When culturing the petite mutant of the strain ∆yku70 at a restrictive temperature of 37 оC for 7 days, the formation of viable subpopulations was detected, which can overcome the arrest of the cell cycle in the G2 / M phase. Further analysis of the isolated clones of adaptive regrowth showed that they differ in cell survival at restrictive temperature, resistance to UV radiation and the ability to form adaptive regrowth on colonies. In the analysis of microsatellite repeats in adaptive regrowth clones, no manifestations of instability of the studied sequences were detected.

Inhibition of DNA damage response at telomeres improves the detrimental phenotypes of Hutchinson\u2013Gilford Progeria Syndrome

Hutchinson–Gilford progeria syndrome (HGPS) is a genetic disorder characterized by premature aging features. Cells from HGPS patients express progerin, a truncated form of Lamin A, which perturbs cellular homeostasis leading to nuclear shape alterations, genome instability, heterochromatin loss, telomere dysfunction and premature entry into cellular senescence. Recently, we reported that telomere dysfunction induces the transcription of telomeric non-coding RNAs (tncRNAs) which control the DNA damage response (DDR) at dysfunctional telomeres. Here we show that progerin-induced telomere dysfunction induces the transcription of tncRNAs. Their functional inhibition by sequence-specific telomeric antisense oligonucleotides (tASOs) prevents full DDR activation and premature cellular senescence in various HGPS cell systems, including HGPS patient fibroblasts. We also show in vivo that tASO treatment significantly enhances skin homeostasis and lifespan in a transgenic HGPS mouse model. In summary, our results demonstrate an important role for telomeric DDR activation in HGPS progeroid detrimental phenotypes in vitro and in vivo.

Reciprocal interactions between mtDNA and lifespan control in budding yeast.

Loss of mitochondrial DNA (mtDNA) results in loss of mitochondrial respiratory activity, checkpoint-regulated inhibition of cell cycle progression, defects in growth, and nuclear genome instability. However, after several generations, yeast cells can adapt to the loss of mtDNA. During this adaptation, rho0 cells, which have no mtDNA, exhibit increased growth rates and nuclear genome stabilization. Here, we report that an immediate response to loss of mtDNA is a decrease in replicative lifespan (RLS). Moreover, we find that adapted rho0 cells bypass the mtDNA inheritance checkpoint, exhibit increased mitochondrial function, and undergo an increase in RLS as they adapt to the loss of mtDNA. Transcriptome analysis reveals that metabolic reprogramming to compensate for defects in mitochondrial function is an early event during adaptation and that up-regulation of stress response genes occurs later in the adaptation process. We also find that specific subtelomeric genes are silenced during adaptation to loss of mtDNA. Moreover, we find that deletion of SIR3, a subtelomeric gene silencing protein, inhibits silencing of subtelomeric genes associated with adaptation to loss of mtDNA, as well as adaptation-associated increases in mitochondrial function and RLS extension.

Age is in the nucleus.

Nuclear DNA damage has detrimental effects on cellular homoeostasis and accelerates the ageing process. A new study causally links error-prone mitochondrial replication to increased nuclear DNA damage, thus suggesting that the hallmarks of ageing are associated with nuclear genome instability, a potential unifying denominator in the ageing process.

Nuclear Genomic Instability and Aging.

The nuclear genome decays as organisms age. Numerous studies demonstrate that the burden of several classes of DNA lesions is greater in older mammals than in young mammals. More challenging is proving this is a cause rather than a consequence of aging. The DNA damage theory of aging, which argues that genomic instability plays a causal role in aging, has recently gained momentum. Support for this theory stems partly from progeroid syndromes in which inherited defects in DNA repair increase the burden of DNA damage leading to accelerated aging of one or more organs. Additionally, growing evidence shows that DNA damage accrual triggers cellular senescence and metabolic changes that promote a decline in tissue function and increased susceptibility to age-related diseases. Here, we examine multiple lines of evidence correlating nuclear DNA damage with aging. We then consider how, mechanistically, nuclear genotoxic stress could promote aging. We conclude that the evidence, in toto, supports a role for DNA damage as a nidus of aging.

Perturbed autophagy and DNA repair converge to promote neurodegeneration in amyotrophic lateral sclerosis and dementia.

Maintaining genomic stability constitutes a major challenge facing cells. DNA breaks can arise from direct oxidative damage to the DNA backbone, the inappropriate activities of endogenous enzymes such as DNA topoisomerases, or due to transcriptionally-derived RNA/DNA hybrids (R-loops). The progressive accumulation of DNA breaks has been linked to several neurological disorders. Recently, however, several independent studies have implicated nuclear and mitochondrial genomic instability, perturbed co-transcriptional processing, and impaired cellular clearance pathways as causal and intertwined mechanisms underpinning neurodegeneration. Here, we discuss this emerging paradigm in the context of amyotrophic lateral sclerosis and frontotemporal dementia, and outline how this knowledge paves the way to novel therapeutic interventions.

Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol.

Human PrimPol is a DNA primase/polymerase involved in DNA damage tolerance and prevents nuclear genome instability. PrimPol is also localized to the mitochondria, but its precise function in mitochondrial DNA maintenance has remained elusive. PrimPol works both as a translesion (TLS) polymerase and as the primase that restarts DNA replication after a lesion. However, the observed biochemical activities of PrimPol vary considerably between studies as a result of different reaction conditions used. To reveal the effects of reaction composition on PrimPol DNA polymerase activity, we tested the polymerase activity in the presence of various buffer agents, salt concentrations, pH values and metal cofactors. Additionally, the enzyme stability was analyzed under various conditions. We demonstrate that the reaction buffer with pH 6–6.5, low salt concentrations and 3 mM Mg2+ or 0.3–3 mM Mn2+ cofactor ions supports the highest DNA polymerase activity of human PrimPol in vitro. The DNA polymerase activity of PrimPol was found to be stable after multiple freeze-thaw cycles and prolonged protein incubation on ice. However, rapid heat-inactivation of the enzyme was observed at 37ºC. We also for the first time describe the purification of human PrimPol from a human cell line and compare the benefits of this approach to the expression in Escherichia coli and in Saccharomyces cerevisiae cells. Our results show that active PrimPol can be purified from E. coli and human suspension cell line in high quantities and that the activity of the purified enzyme is similar in both expression systems. Conversely, the yield of full-length protein expressed in S. cerevisiae was considerably lower and this system is therefore not recommended for expression of full-length recombinant human PrimPol.