Mitochondrial DNA metabolism is coupled with 20S proteasome function via regulation of deoxyribonucleotide homeostasis in Saccharomyces cerevisiae

The synthesis of mitochondrial DNA (mtDNA) is not coupled with cell cycle. Previous studies have shown that the size of deoxyribonucleoside triphosphate (dNTP) pools plays an important role in regulating mtDNA replication and amplification. In yeast, dNTPs are synthesized by the cytosolic ribonucleotide reductase (RNR). It is currently poorly understood as to how RNR activity is regulated in non-dividing or quiescent cells to finely tune mtDNA metabolism to cope with different metabolic states. Here, we show that defect in the 20S proteasome drastically destabilizes mtDNA. The mtDNA instability phenotype in 20S proteasome mutants is suppressed by overexpression of RNR3 or by the deletion of SML1, encoding a minor catalytic subunit and an intrinsic inhibitor of RNR respectively. We found that Sml1 is stabilized in the 20S proteasomal mutants, suggesting that 20S affects mtDNA stability by stabilizing Sml1. Interestingly, defect in the regulatory 19S proteasomal function has only subtle effect on mtDNA stability, supporting a role of the 20S proteasome in dNTP homeostasis independent of 19S. Finally, we found that when cells are transitioned from glycolytic to oxidative growth, Sml1 level is reduced in a 20S-dependent manner. In summary, our study establishes a link between cellular proteostasis and mtDNA metabolism through the regulation of dNTP homeostasis. We propose that increased degradation of Sml1 by the 20S proteasome under respiratory conditions provides a mechanism to stimulate dNTP synthesis and promote mtDNA amplification.

RRM2 Improves Cardiomyocyte Proliferation after Myocardial Ischemia Reperfusion Injury through the Hippo-YAP Pathway

Objective. Ribonucleotide reductase M2 (RRM2) as an enzyme that catalyzes the deoxyreduction of nucleosides to deoxyribonucleoside triphosphate (dNTP) has been extensively studied, and it plays a crucial role in regulating cell proliferation. However, its role in ischemia-reperfusion injury (I/RI) is still unclear. Methods. SD rats were used as the research object to detect the expression of RRM2 in the myocardium by constructing an I/RI model. At the same time, primary SD neonatal rat cardiomyocytes were extracted, and hypoxia/reoxygenation (H/R) treatment simulated the I/RI model. Using transfection technology to overexpress RRM2 in cardiomyocytes, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was used to detect the expression of RRM2, Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability, and immunofluorescence staining was used to detect Ki67 and EdU-positive cells. Western blot (WB) technology was used to detect YAP and its phosphorylation expression. Results. qRT-PCR results indicated that the expression of RRM2 was inhibited in the model group, and cardiomyocytes overexpressing RRM2 can obviously promote the proliferation of primary cardiomyocytes and improve the damage of cardiac structure and function caused by I/R. At the same time, RRM2 can promote the increase of YAP protein expression and the increase of Cyclin D1 mRNA expression. Conclusion. RRM2 expression was downregulated in myocardial tissue with I/R. After overexpression of RRM2, cardiomyocyte proliferation was upregulated and the Hippo-YAP signaling pathway was activated.

A Yeast-Based Repurposing Approach for the Treatment of Mitochondrial DNA Depletion Syndromes Led to the Identification of Molecules Able to Modulate the dNTP Pool

Mitochondrial DNA depletion syndromes (MDS) are clinically heterogenous and often severe diseases, characterized by a reduction of the number of copies of mitochondrial DNA (mtDNA) in affected tissues. In the context of MDS, yeast has proved to be both an excellent model for the study of the mechanisms underlying mitochondrial pathologies and for the discovery of new therapies via high-throughput assays. Among the several genes involved in MDS, it has been shown that recessive mutations in MPV17 cause a hepatocerebral form of MDS and Navajo neurohepatopathy. MPV17 encodes a non selective channel in the inner mitochondrial membrane, but its physiological role and the nature of its cargo remains elusive. In this study we identify ten drugs active against MPV17 disorder, modelled in yeast using the homologous gene SYM1. All ten of the identified molecules cause a concomitant increase of both the mitochondrial deoxyribonucleoside triphosphate (mtdNTP) pool and mtDNA stability, which suggests that the reduced availability of DNA synthesis precursors is the cause for the mtDNA deletion and depletion associated with Sym1 deficiency. We finally evaluated the effect of these molecules on mtDNA stability in two other MDS yeast models, extending the potential use of these drugs to a wider range of MDS patients.

Nucleotide Pool Imbalance and Antibody Gene Diversification

The availability and adequate balance of deoxyribonucleoside triphosphate (dNTP) is an important determinant of both the fidelity and the processivity of DNA polymerases. Therefore, maintaining an optimal balance of the dNTP pool is critical for genomic stability in replicating and quiescent cells. Since DNA synthesis is required not only in genomic replication but also in DNA damage repair and recombination, the abnormalities in the dNTP pool affect a wide range of chromosomal activities. The generation of antibody diversity relies on antigen-independent V(D)J recombination, as well as antigen-dependent somatic hypermutation and class switch recombination. These processes involve diverse sets of DNA polymerases, which are affected by the dNTP pool imbalances. This review discusses the role of the optimal dNTP pool balance in the diversification of antibody encoding genes.

SAMHD1 in cancer: curse or cure?

Human sterile α motif and HD domain-containing protein 1 (SAMHD1), originally described as the major cellular deoxyribonucleoside triphosphate triphosphohydrolase (dNTPase) balancing the intracellular deoxynucleotide (dNTP) pool, has come recently into focus of cancer research. As outlined in this review, SAMHD1 has been reported to be mutated in a variety of cancer types and the expression of SAMHD1 is dysregulated in many cancers. Therefore, SAMHD1 is regarded as a tumor suppressor in certain tumors. Moreover, it has been proposed that SAMHD1 might fulfill the requirements of a driver gene in tumor development or might promote a so-called mutator phenotype. Besides its role as a dNTPase, several novel cellular functions of SAMHD1 have come to light only recently, including a role as negative regulator of innate immune responses and as facilitator of DNA end resection during DNA replication and repair. Therefore, SAMHD1 can be placed at the crossroads of various cellular processes. The present review summarizes the negative role of SAMHD1 in chemotherapy sensitivity, highlights reported SAMHD1 mutations found in various cancer types, and aims to discuss functional consequences as well as underlying mechanisms of SAMHD1 dysregulation potentially involved in cancer development.

Complex Mutation Profiles in Mismatch Repair and Ribonucleotide Reductase Mutations Reveal Novel Repair Substrate Specificity of MutS Homolog (MSH) Complexes

Determining mutation signatures is standard for understanding the etiology of human tumors and informing cancer treatment. Multiple determinants of DNA replication fidelity prevent mutagenesis that leads to carcinogenesis, including regulation of free deoxyribonucleoside triphosphate (dNTP) pools and the repair of base mismatches. We utilized a targeted deep-sequencing approach to determine mutational signatures associated with mismatch repair (MMR) pathway defects. By combining rnr1 and msh mutations to increase dNTP levels and alter the mutational load, we observed previously unreported specificities of Msh2-Msh3 and Msh2-Msh6. Msh2-Msh3 is uniquely able to direct repair of G/C single base deletions in GC runs, while Msh2-Msh6 specifically directs repair of substitutions at G/C dinucleotides. We also identified broader sequence contexts that influence variant profiles in different genetic backgrounds and found that there was not necessarily a simple additive relationship of mutation profiles in double mutants. Our results have implications for interpreting mutation signatures from human tumors, particularly when MMR is defective.

Multiple deprotonation paths of the nucleophile 3′-OH in the DNA synthesis reaction

DNA synthesis by polymerases is essential for life. Deprotonation of the nucleophile 3′-OH is thought to be the obligatory first step in the DNA synthesis reaction. We have examined each entity surrounding the nucleophile 3′-OH in the reaction catalyzed by human DNA polymerase (Pol) η and delineated the deprotonation process by combining mutagenesis with steady-state kinetics, high-resolution structures of in crystallo reactions, and molecular dynamics simulations. The conserved S113 residue, which forms a hydrogen bond with the primer 3′-OH in the ground state, stabilizes the primer end in the active site. Mutation of S113 to alanine destabilizes primer binding and reduces the catalytic efficiency. Displacement of a water molecule that is hydrogen bonded to the 3′-OH using the 2′-OH of a ribonucleotide or 2′-F has little effect on catalysis. Moreover, combining the S113A mutation with 2′-F replacement, which removes two potential hydrogen acceptors of the 3′-OH, does not reduce the catalytic efficiency. We conclude that the proton can leave the O3′ via alternative paths, supporting the hypothesis that binding of the third Mg2+ initiates the reaction by breaking the α–β phosphodiester bond of an incoming deoxyribonucleoside triphosphate (dNTP).

Mechanisms of Chronic Fialuridine Hepatotoxicity as Revealed in Primary Human Hepatocyte Spheroids

Drug hepatotoxicity is often delayed in onset. An exemplar case is the chronic nature of fialuridine hepatotoxicity, which resulted in the deaths of several patients in clinical trials as preclinical studies failed to identify this human-specific hepatotoxicity. Conventional preclinical in vitro models are mainly designed to evaluate the risk of acute drug toxicity. Here, we evaluated the utility of 3D spheroid cultures of primary human hepatocytes (PHHs) to assess chronic drug hepatotoxicity events using fialuridine as an example. Fialuridine toxicity was only detectable after 7 days of repeated exposure. Clinical manifestations, including reactive oxygen species formation, lipid accumulation, and induction of apoptosis, were readily identified. Silencing the expression or activity of the human equilibrative nucleoside transporter 1 (ENT1), implicated in the mitochondrial transport of fialuridine, modestly protected PHH spheroids from fialuridine toxicity. Interference with the phosphorylation of fialuridine into the active triphosphate metabolites by silencing of thymidine kinase 2 (TK2) provided substantial protection, whereas simultaneous silencing of ENT1 and TK2 provided near-complete protection. Fialuridine-induced mitochondrial dysfunction was suggested by a decrease in the expression of mtDNA-encoded genes, which correlated with the onset of toxicity and was prevented under the simultaneous silencing of ENT1 and TK2. Furthermore, interference with the expression or activity of ribonucleotide reductase (RNR), which is critical to deoxyribonucleoside triphosphate (dNTP) pool homeostasis, resulted in selective potentiation of fialuridine toxicity. Our findings demonstrate the translational applicability of the PHH 3D spheroid model for assessing drug hepatotoxicity events which manifest only under chronic exposure conditions.

A dual role for SAMHD1 in regulating HBV cccDNA and RT-dependent particle genesis

Chronic hepatitis B is one of the world’s unconquered diseases with more than 240 million infected subjects at risk of developing liver disease and hepatocellular carcinoma. Hepatitis B virus reverse transcribes pre-genomic RNA to relaxed circular DNA (rcDNA) that comprises the infectious particle. To establish infection of a naïve target cell, the newly imported rcDNA is repaired by host enzymes to generate covalently closed circular DNA (cccDNA), which forms the transcriptional template for viral replication. SAMHD1 is a component of the innate immune system that regulates deoxyribonucleoside triphosphate levels required for host and viral DNA synthesis. Here, we show a positive role for SAMHD1 in regulating cccDNA formation, where KO of SAMHD1 significantly reduces cccDNA levels that was reversed by expressing wild-type but not a mutated SAMHD1 lacking the nuclear localization signal. The limited pool of cccDNA in infectedSamhd1KO cells is transcriptionally active, and we observed a 10-fold increase in newly synthesized rcDNA-containing particles, demonstrating a dual role for SAMHD1 to both facilitate cccDNA genesis and to restrict reverse transcriptase-dependent particle genesis.

Protected 2′-deoxyribonucleoside triphosphate building blocks for the photocaging of epigenetic 5-(hydroxymethyl)cytosine in DNA

2′-Deoxyribonucleoside triphosphates containing 5-(hydroxymethyl)cytosine protected with photocleavable groups were prepared and studied as substrates for the enzymatic synthesis of DNA containing a photocaged epigenetic 5hmC base.