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G3 (Bethesda, Md.) Apr 2024All animals must maintain genome and proteome integrity, especially when experiencing endogenous or exogenous stress. To cope, organisms have evolved sophisticated and...
All animals must maintain genome and proteome integrity, especially when experiencing endogenous or exogenous stress. To cope, organisms have evolved sophisticated and conserved response systems: unfolded protein responses (UPRs) ensure proteostasis, while DNA damage responses (DDRs) maintain genome integrity. Emerging evidence suggests that UPRs and DDRs crosstalk, but this remains poorly understood. Here, we demonstrate that depletion of the DNA primases pri-1 or pri-2, which synthesize RNA primers at replication forks and whose inactivation causes DNA damage, activates the UPR of the endoplasmic reticulum (UPR-ER) in Caenorhabditis elegans, with especially strong activation in the germline. We observed activation of both the inositol-requiring-enzyme 1 (ire-1) and the protein kinase RNA-like endoplasmic reticulum kinase (pek-1) branches of the (UPR-ER). Interestingly, activation of the (UPR-ER) output gene heat shock protein 4 (hsp-4) was partially independent of its canonical activators, ire-1 and X-box binding protein (xbp-1), and instead required the third branch of the (UPR-ER), activating transcription factor 6 (atf-6), suggesting functional redundancy. We further found that primase depletion specifically induces the (UPR-ER), but not the distinct cytosolic or mitochondrial UPRs, suggesting that primase inactivation causes compartment-specific rather than global stress. Functionally, loss of ire-1 or pek-1 sensitizes animals to replication stress caused by hydroxyurea. Finally, transcriptome analysis of pri-1 embryos revealed several deregulated processes that could cause (UPR-ER) activation, including protein glycosylation, calcium signaling, and fatty acid desaturation. Together, our data show that the (UPR-ER), but not other UPRs, responds to replication fork stress and that the (UPR-ER) is required to alleviate this stress.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; DNA Primase; Unfolded Protein Response; Cell Cycle Proteins; DNA Damage; Endoplasmic Reticulum; Endoplasmic Reticulum Stress
PubMed: 38267027
DOI: 10.1093/g3journal/jkae017 -
Nature Communications Jan 2024
PubMed: 38225289
DOI: 10.1038/s41467-024-44904-0 -
International Journal of Molecular... Dec 2023PrimPol is a DNA primase/polymerase from the Archaeo-Eukaryotic Primase (AEP) superfamily that enables the progression of stalled replication forks by synthesizing DNA...
PrimPol is a DNA primase/polymerase from the Archaeo-Eukaryotic Primase (AEP) superfamily that enables the progression of stalled replication forks by synthesizing DNA primers ahead of blocking lesions or abnormal structures in the ssDNA template. PrimPol's active site is formed by three AEP-conserved motifs: A, B and C. Motifs A and C of human PrimPol (PrimPol) harbor the catalytic residues (Asp, Glu, Asp) acting as metal ligands, whereas motif B includes highly conserved residues (Lys, Ser and His), which are postulated to stabilize 3' incoming deoxynucleotides (dNTPs). Additionally, other putative nucleotide ligands are situated close to motif C: Lys, almost invariant in the whole AEP superfamily, and Lys, specifically conserved in eukaryotic PrimPols. Here, we demonstrate that His is absolutely essential for 3'dNTP binding and, hence, for both primase and polymerase activities of PrimPol, whereas Ser and Lys are crucial for the dimer synthesis initiation step during priming, but dispensable for subsequent dNTP incorporation on growing primers. Conversely, the elimination of Lys does not affect the overall primase function; however, it is required for damage avoidance via primer-template realignments. Finally, Lys is identified as an extra anchor residue to stabilize the 3' incoming dNTP. Collectively, these results demonstrate that individual ligands modulate the stabilization of 3' incoming dNTPs to optimize DNA primer synthesis efficiency during initiation and primer maturation.
Topics: Humans; DNA Primase; Nucleotidyltransferases; Catalysis; Cognition; DNA Primers; Nucleotides; DNA-Directed DNA Polymerase; Multifunctional Enzymes
PubMed: 38203225
DOI: 10.3390/ijms25010051 -
Nature Structural & Molecular Biology Jan 2024The Mpox pandemic, caused by the Mpox virus (or monkeypox virus, MPXV), has gained global attention. The D5 protein, a putative helicase-primase found in MPXV, plays a...
The Mpox pandemic, caused by the Mpox virus (or monkeypox virus, MPXV), has gained global attention. The D5 protein, a putative helicase-primase found in MPXV, plays a vital role in viral replication and genome uncoating. Here we determined multiple cryo-EM structures of full-length hexameric D5 in diverse states. These states were captured during ATP hydrolysis while moving along the single-stranded DNA (ssDNA) track. Through comprehensive structural analysis combined with the helicase activity system, we revealed that when the primase domain is truncated or the interaction between the primase and helicase domains is disrupted, the double-stranded DNA (dsDNA) unwinds into ssDNA, suggesting a critical regulatory role of the primase domain. Two transition states bound with ssDNA substrate during unwinding reveals that two ATP molecules were consumed to drive DNA moving forward two nucleotides. Collectively, our findings shed light on the molecular mechanism that links ATP hydrolysis to the DNA unwinding in poxviruses.
Topics: DNA Primase; Monkeypox virus; DNA Helicases; DNA; DNA, Single-Stranded; Adenosine Triphosphate
PubMed: 38177671
DOI: 10.1038/s41594-023-01142-0 -
Nature Communications Jan 2024Transcription-replication conflicts (TRCs), especially Head-On TRCs (HO-TRCs) can introduce R-loops and DNA damage, however, the underlying mechanisms are still largely...
Transcription-replication conflicts (TRCs), especially Head-On TRCs (HO-TRCs) can introduce R-loops and DNA damage, however, the underlying mechanisms are still largely unclear. We previously identified a chloroplast-localized RNase H1 protein AtRNH1C that can remove R-loops and relax HO-TRCs for genome integrity. Through the mutagenesis screen, we identify a mutation in chloroplast-localized primase ATH that weakens the binding affinity of DNA template and reduces the activities of RNA primer synthesis and delivery. This slows down DNA replication, and reduces competition of transcription-replication, thus rescuing the developmental defects of atrnh1c. Strand-specific DNA damage sequencing reveals that HO-TRCs cause DNA damage at the end of the transcription unit in the lagging strand and overexpression of ATH can boost HO-TRCs and exacerbates DNA damage. Furthermore, mutation of plastid DNA polymerase Pol1A can similarly rescue the defects in atrnh1c mutants. Taken together these results illustrate a potentially conserved mechanism among organisms, of which the primase activity can promote the occurrence of transcription-replication conflicts leading to HO-TRCs and genome instability.
Topics: DNA Primase; DNA Replication; DNA-Directed DNA Polymerase; DNA Damage; Mutation
PubMed: 38168108
DOI: 10.1038/s41467-023-44443-0 -
Molecular Informatics Mar 2024Tuberculosis (TB) is the second leading cause of mortality after COVID-19, with a global death toll of 1.6 million in 2021. The escalating situation of drug-resistant...
Tuberculosis (TB) is the second leading cause of mortality after COVID-19, with a global death toll of 1.6 million in 2021. The escalating situation of drug-resistant forms of TB has threatened the current TB management strategies. New therapeutics with novel mechanisms of action are urgently required to address the current global TB crisis. The essential mycobacterial primase DnaG with no structural homology to homo sapiens presents itself as a good candidate for drug targeting. In the present study, Mitoxantrone and Vapreotide, two FDA-approved drugs, were identified as potential anti-mycobacterial agents. Both Mitoxantrone and Vapreotide exhibit a strong Minimum Inhibitory Concentration (MIC) of ≤25μg/ml against both the virulent (M.tb-H37Rv) and avirulent (M.tb-H37Ra) strains of M.tb. Extending the validations further revealed the inhibitory potential drugs in ex vivo conditions. Leveraging the computational high-throughput multi-level docking procedures from the pool of ~2700 FDA-approved compounds, Mitoxantrone and Vapreotide were screened out as potential inhibitors of DnaG. Extensive 200 ns long all-atoms molecular dynamic simulation of DnaGDrugs complexes revealed that both drugs bind strongly and stabilize the DnaG during simulations. Reduced solvent exposure and confined motions of the active centre of DnaG upon complexation with drugs indicated that both drugs led to the closure of the active site of DnaG. From this study's findings, we propose Mitoxantrone and Vapreotide as potential anti-mycobacterial agents, with their novel mechanism of action against mycobacterial DnaG.
Topics: Humans; Mycobacterium tuberculosis; Antitubercular Agents; DNA Primase; Mitoxantrone; Somatostatin
PubMed: 38123523
DOI: 10.1002/minf.202300284 -
Biochemistry. Biokhimiia Nov 2023Human DNA primase/polymerase PrimPol synthesizes DNA primers de novo after replication fork stalling at the sites of DNA damage, thus contributing to the DNA damage...
Human DNA primase/polymerase PrimPol synthesizes DNA primers de novo after replication fork stalling at the sites of DNA damage, thus contributing to the DNA damage tolerance. The role of PrimPol in response to the different types of DNA damage is poorly understood. We knocked out the PRIMPOL gene in the lung carcinoma A549 cell line and characterized the response of the obtained cells to the DNA damage caused by hydrogen peroxide, methyl methanesulfonate (MMS), cisplatin, bleomycin, and ionizing radiation. The PRIMPOL knockout reduced the number of proliferating cells and cells in the G2 phase after treatment with MMS and caused a more pronounced delay of the S phase in the cisplatin-treated cells. Ionizing radiation at a dose of 10 Gy significantly increased the content of apoptotic cells among the PRIMPOL-deficient cells, while the proportion of cells undergoing necroptosis increased in both parental and knockout cells at any radiation dose. The viability of PRIMPOL-deficient cells upon the hydrogen peroxide-induced oxidative stress increased compared to the control cells, as determined by the methyl tetrazolium (MTT) assay. The obtained data indicate the involvement of PRIMPOL in the modulation of adaptive cell response to various types of genotoxic stress.
Topics: Humans; DNA-Directed DNA Polymerase; A549 Cells; Cisplatin; Hydrogen Peroxide; DNA Replication; DNA Damage; Adenocarcinoma of Lung; DNA Primase; Multifunctional Enzymes
PubMed: 38105210
DOI: 10.1134/S0006297923110214 -
Journal of Biotechnology Jan 2024DNA damage tolerance (DDT) pathways mitigate the effects of DNA damage during replication by rescuing the replication fork stalled at a DNA lesion or other barriers and... (Review)
Review
DNA damage tolerance (DDT) pathways mitigate the effects of DNA damage during replication by rescuing the replication fork stalled at a DNA lesion or other barriers and also repair discontinuities left in the newly replicated DNA. From yeast to mammalian cells, RAD18-regulated translesion synthesis (TLS) and template switching (TS) represent the dominant pathways of DDT. Monoubiquitylation of the polymerase sliding clamp PCNA by HRAD6A-B/RAD18, an E2/E3 protein pair, enables the recruitment of specialized TLS polymerases that can insert nucleotides opposite damaged template bases. Alternatively, the subsequent polyubiquitylation of monoubiquitin-PCNA by Ubc13-Mms2 (E2) and HLTF or SHPRH (E3) can lead to the switching of the synthesis from the damaged template to the undamaged newly synthesized sister strand to facilitate synthesis past the lesion. When immediate TLS or TS cannot occur, gaps may remain in the newly synthesized strand, partly due to the repriming activity of the PRIMPOL primase, which can be filled during the later phases of the cell cycle. The first part of this review will summarize the current knowledge about RAD18-dependent DDT pathways, while the second part will offer a molecular toolkit for the identification and characterization of the cellular functions of a DDT protein. In particular, we will focus on advanced techniques that can reveal single-stranded and double-stranded DNA gaps and their repair at the single-cell level as well as monitor the progression of single replication forks, such as the specific versions of the DNA fiber and comet assays. This collection of methods may serve as a powerful molecular toolkit to monitor the metabolism of gaps, detect the contribution of relevant pathways and molecular players, as well as characterize the effectiveness of potential inhibitors.
Topics: Animals; DNA Replication; Proliferating Cell Nuclear Antigen; DNA Damage; DNA; Saccharomyces cerevisiae; DNA Repair; Mammals; DNA-Binding Proteins; Saccharomyces cerevisiae Proteins
PubMed: 38072328
DOI: 10.1016/j.jbiotec.2023.12.001 -
Clinical Nutrition ESPEN Dec 2023The presented review is an updating of Iron metabolism in context of normal physiology and pathological phases. Iron is one of the vital elements in humans and... (Review)
Review
PURPOSE (BACKGROUND)
The presented review is an updating of Iron metabolism in context of normal physiology and pathological phases. Iron is one of the vital elements in humans and associated into proteins as a component of heme (e.g. hemoglobin, myoglobin, cytochromes proteins, myeloperoxidase, nitric oxide synthetases), iron sulfur clusters (e.g. respiratory complexes I-III, coenzyme Q10, mitochondrial aconitase, DNA primase), or other functional groups (e.g. hypoxia inducible factor prolyl hydroxylases). All these entire iron-containing proteins ar e needed for vital cellular and organismal functions together with oxygen transport, mitochondrial respiration, intermediary and xenobiotic metabolism, nucleic acid replication and repair, host defense, and cell signaling.
METHODS (METABOLIC STRATEGIES)
Cells have developed metabolic strategies to import and employ iron safely. Regulatory process of iron uptake, storage, intracellular trafficking and utilization is vital for the maintenance of cellular iron homeostasis. Cellular iron utilization and intracellular iron trafficking pathways are not well established and very little knowledge about this. The predominant organs, which are associated in the metabolism of iron, are intestine, liver, bone marrow and spleen. Iron is conserved, recycled and stored. The reduced bioavailability of iron in humans has developed extremely efficient mechanisms for iron conservation. Prominently, the losses of iron cannot considerably enhance through physiologic mechanisms, even if iron intake and stores become excessive. Loss of iron is balanced or maintained from dietary sources.
RESULTS (OUTCOMES)
Numerous physiological abnormalities are associated with impaired iron metabolism. These abnormalities are appeared in the form of several diseases. There are duodenal ulcer, inflammatory bowel disease, sideroblastic anaemia, congenital dyserythropoietic anemias and low-grade myelodysplastic syndromes. Hereditary hemochromatosis and anaemia are two chronic diseases, which are responsible for disturbing the iron metabolism in various tissues, including the spleen and the intestine. Impairment in hepatic hepcidin synthesis is responsible for chronic liver disease, which is grounding from alcoholism or viral hepatitis. This condition directs to iron overload that can cause further hepatic damage. Iron has important role in several infectious diseases are tuberculosis, malaria trypanosomatid diseases and acquired immunodeficiency syndrome (AIDS). Iron is also associated with Systemic lupus erythematosus [SLE], cancer, Alzheimer's disease (AD) and post-traumatic epilepsy.
CONCLUSION
Recently, numerous research studies are gradually more dedicated in the field of iron metabolism, but a number of burning questions are still waiting for answer. Cellular iron utilization and intracellular iron trafficking pathways are not well established and very little knowledge about this. Increased information of the physiology of iron homeostasis will support considerate of the pathology of iron disorders and also make available the support to advance treatment.
Topics: Humans; Iron; Hemochromatosis; Iron Overload; Homeostasis; Liver Diseases
PubMed: 38057018
DOI: 10.1016/j.clnesp.2023.10.006 -
Nucleic Acids Research Jan 2024The primase/polymerase PRIMPOL restarts DNA synthesis when replication is arrested by template impediments. However, we do not have a comprehensive view of how...
The primase/polymerase PRIMPOL restarts DNA synthesis when replication is arrested by template impediments. However, we do not have a comprehensive view of how PRIMPOL-dependent repriming integrates with the main pathways of damage tolerance, REV1-dependent 'on-the-fly' lesion bypass at the fork and PCNA ubiquitination-dependent post-replicative gap filling. Guided by genome-wide CRISPR/Cas9 screens to survey the genetic interactions of PRIMPOL in a non-transformed and p53-proficient human cell line, we find that PRIMPOL is needed for cell survival following loss of the Y-family polymerases REV1 and POLη in a lesion-dependent manner, while it plays a broader role in promoting survival of cells lacking PCNA K164-dependent post-replicative gap filling. Thus, while REV1- and PCNA K164R-bypass provide two layers of protection to ensure effective damage tolerance, PRIMPOL is required to maximise the effectiveness of the interaction between them. We propose this is through the restriction of post-replicative gap length provided by PRIMPOL-dependent repriming.
Topics: Humans; DNA Damage; DNA Primase; DNA Replication; Multifunctional Enzymes; Proliferating Cell Nuclear Antigen; DNA-Directed DNA Polymerase
PubMed: 37971291
DOI: 10.1093/nar/gkad1054