-
Viruses Mar 2024CrAss-like phages play an important role in maintaining ecological balance in the human intestinal microbiome. However, their genetic diversity and lifestyle are still...
CrAss-like phages play an important role in maintaining ecological balance in the human intestinal microbiome. However, their genetic diversity and lifestyle are still insufficiently studied. In this study, a novel CrAssE-Sib phage genome belonging to the epsilon crAss-like phage genomes was found. Comparative analysis indicated that epsilon crAss-like phages are divided into two putative genera, which were proposed to be named and ; CrAssE-Sib belongs to the former. The crAssE-Sib genome contains a diversity-generating retroelement (DGR) cassette with all essential elements, including the reverse transcriptase (RT) and receptor binding protein (RBP) genes. However, this RT contains the GxxxSP motif in its fourth domain instead of the usual GxxxSQ motif found in all known phage and bacterial DGRs. RBP encoded by CrAssE-Sib and other has an unusual structure, and no similar phage proteins were found. In addition, crAssE-Sib and other encode conserved prophage repressor and anti-repressors that could be involved in lysogenic-to-lytic cycle switches. Notably, DNA primase sequences of epsilon crAss-like phages are not included in the monophyletic group formed by the DNA primases of all other crAss-like phages. Therefore, epsilon crAss-like phage substantially differ from other crAss-like phages, indicating the need to classify these phages into a separate family.
Topics: Genome, Viral; Bacteriophages; Phylogeny; Viral Proteins; Retroelements; Genetic Variation; Prophages; DNA, Viral; DNA Primase; Genomics; RNA-Directed DNA Polymerase
PubMed: 38675856
DOI: 10.3390/v16040513 -
Nucleic Acids Research Jun 2024Replication repriming by the specialized primase-polymerase PRIMPOL ensures the continuity of DNA synthesis during replication stress. PRIMPOL activity generates...
Replication repriming by the specialized primase-polymerase PRIMPOL ensures the continuity of DNA synthesis during replication stress. PRIMPOL activity generates residual post-replicative single-stranded nascent DNA gaps, which are linked with mutagenesis and chemosensitivity in BRCA1/2-deficient models, and which are suppressed by replication fork reversal mediated by the DNA translocases SMARCAL1 and ZRANB3. Here, we report that the MRE11 regulator MRNIP limits the prevalence of PRIMPOL and MRE11-dependent ssDNA gaps in cells in which fork reversal is perturbed either by treatment with the PARP inhibitor Olaparib, or by depletion of SMARCAL1 or ZRANB3. MRNIP-deficient cells are sensitive to PARP inhibition and accumulate PRIMPOL-dependent DNA damage, supportive of a pro-survival role for MRNIP linked to the regulation of gap prevalence. In MRNIP-deficient cells, post-replicative gap filling is driven in S-phase by UBC13-mediated template switching involving REV1 and the TLS polymerase Pol-ζ. Our findings represent the first report of modulation of post-replicative ssDNA gap dynamics by a direct MRE11 regulator.
PubMed: 38917325
DOI: 10.1093/nar/gkae546 -
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 -
Journal of Molecular Biology May 2024PrimPol is a human DNA primase-polymerase which restarts DNA synthesis beyond DNA lesions and non-B DNA structures blocking replication. Disfunction of PrimPol in cells...
PrimPol is a human DNA primase-polymerase which restarts DNA synthesis beyond DNA lesions and non-B DNA structures blocking replication. Disfunction of PrimPol in cells leads to slowing of DNA replication rates in mitochondria and nucleus, accumulation of chromosome aberrations, cell cycle delay, and elevated sensitivity to DNA-damaging agents. A defective PrimPol has been suggested to be associated with the development of ophthalmic diseases, elevated mitochondrial toxicity of antiviral drugs and increased cell resistance to chemotherapy. Here, we describe a rare missense PrimPol variant V102A with altered biochemical properties identified in patients suffering from ovarian and cervical cancer. The Val102 to Ala substitution dramatically reduced both the primase and DNA polymerase activities of PrimPol as well as specifically decreased its ability to incorporate ribonucleotides. Structural analysis indicates that the V102A substitution can destabilize the hydrophobic pocket adjacent to the active site, affecting dNTP binding and catalysis.
Topics: Female; Humans; Amino Acid Substitution; Catalytic Domain; Crystallography, X-Ray; DNA Primase; DNA Replication; DNA-Directed DNA Polymerase; Models, Molecular; Multifunctional Enzymes; Mutation, Missense; Protein Conformation; Uterine Cervical Neoplasms; Ovarian Neoplasms
PubMed: 38492718
DOI: 10.1016/j.jmb.2024.168542 -
Journal of Molecular Biology Dec 2023To facilitate the eukaryotic repriming pathway of DNA damage tolerance, PrimPol synthesises de novo oligonucleotide primers downstream of polymerase-stalling obstacles....
To facilitate the eukaryotic repriming pathway of DNA damage tolerance, PrimPol synthesises de novo oligonucleotide primers downstream of polymerase-stalling obstacles. These primers enable replicative polymerases to resume synthesis and ensure the timely completion of DNA replication. Initiating synthesis de novo requires the coordination of single-stranded DNA, initiating nucleotides, and metal ions within PrimPol's active site to catalyze the formation of the first phosphodiester bond. Here we examine the interactions between human PrimPol's catalytic domain, nucleotides, and DNA template during each of the various catalytic steps to determine the 'choreography' of primer synthesis, where substrates bind in an ordered manner. Our findings show that the ability of PrimPol to conduct de novo primer synthesis is underpinned by a network of stabilising interactions between the enzyme, template, and nucleotides, as we previously observed for related primase CRISPR-Associated Prim-Pol (CAPP). Together, these findings establish a detailed model for the initiation of DNA synthesis by human PrimPol, which appears highly conserved.
Topics: Humans; Catalytic Domain; DNA Primase; DNA Replication; DNA, Single-Stranded; DNA-Directed DNA Polymerase; Multifunctional Enzymes; Nucleotides
PubMed: 37923120
DOI: 10.1016/j.jmb.2023.168338 -
ACS Nano Aug 2023Direct delivery of therapeutic genes is a promising approach for treating cancers and other diseases. The current human viral vectors, however, suffer from several...
Direct delivery of therapeutic genes is a promising approach for treating cancers and other diseases. The current human viral vectors, however, suffer from several drawbacks, including poor cell-type specificity and difficult large-scale production. The M13 phage provides an alternative vehicle for gene therapy with engineerable specificity, but the low transduction efficiency seriously limits its translational application. In this work, we discovered important factors of cells and phages that greatly influence the phage transduction. The up-regulation of PrimPol or the down-regulation of DMBT1 in cells significantly enhanced the phage transduction efficiency. Furthermore, we found that the phage transduction efficiency was inversely correlated with the phage size. By carefully reconstructing the phage origin with the gene of interest, we designed "TransPhage" with a minimal length and maximal transduction efficiency. We showed that TransPhage successfully transduced the human cells with an excellent efficiency (up to 95%) comparable to or superior to that of the adeno-associated virus vectors. Moreover, we showed that TransPhage's tropism was specific to the cells that overexpress the target antigen, whereas adeno-associated viruses (AAVs) promiscuously infected many cell types. Using TransPhage as a gene therapy vehicle, we invented an NK-cell-mediated immunotherapy in which a membrane-bound fragment crystallizable region was introduced to cancer cells. We showed that the cancer cells expressing the membrane-bound fragment crystallizable (Fc) were effectively killed by CD16+ NK cells through an antibody-dependent cell-mediated cytotoxicity (ADCC)-like mechanism. In the xenograft mouse model, the administration of TransPhage carrying the membrane-bound Fc gene greatly suppressed tumor growth.
Topics: Humans; Mice; Animals; Gene Transfer Techniques; Genetic Vectors; Bacteriophage M13; Genetic Therapy; Killer Cells, Natural; Neoplasms; Calcium-Binding Proteins; DNA-Binding Proteins; Tumor Suppressor Proteins; DNA-Directed DNA Polymerase; DNA Primase; Multifunctional Enzymes
PubMed: 37466994
DOI: 10.1021/acsnano.3c01295 -
BMC Plant Biology Oct 2023The mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically...
BACKGROUND
The mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically studied. A primase-helicase dubbed Twinkle (T7 gp4-like protein with intramitochondrial nucleoid localization) unwinds double-stranded DNA in metazoan mitochondria and plant organelles. Twinkle in plants is a bifunctional enzyme with an active primase module. This contrast with animal Twinkle in which the primase module is inactive. The organellar primase-helicase of Arabidopsis thaliana (AtTwinkle) harbors a primase module (AtPrimase) that consists of an RNA polymerase domain (RPD) and a Zn + + finger domain (ZFD).
RESULTS
Herein, we investigate the mechanisms by which AtTwinkle recognizes its templating sequence and how primer synthesis and coupling to the organellar DNA polymerases occurs. Biochemical data show that the ZFD of the AtPrimase module is responsible for template recognition, and this recognition is achieved by residues N163, R166, and K168. The role of the ZFD in template recognition was also corroborated by swapping the RPDs of bacteriophage T7 primase and AtPrimase with their respective ZFDs. A chimeric primase harboring the ZFD of T7 primase and the RPD of AtPrimase synthesizes ribonucleotides from the T7 primase recognition sequence and conversely, a chimeric primase harboring the ZFD of AtPrimase and the RPD of T7 primase synthesizes ribonucleotides from the AtPrimase recognition sequence. A chimera harboring the RPDs of bacteriophage T7 and the ZBD of AtTwinkle efficiently synthesizes primers for the plant organellar DNA polymerase.
CONCLUSIONS
We conclude that the ZFD is responsible for recognizing a single-stranded sequence and for primer hand-off into the organellar DNA polymerases active site. The primase activity of plant Twinkle is consistent with phylogeny-based reconstructions that concluded that Twinkle´s last eukaryotic common ancestor (LECA) was an enzyme with primase and helicase activities. In plants, the primase domain is active, whereas the primase activity was lost in metazoans. Our data supports the notion that AtTwinkle synthesizes primers at the lagging-strand of the organellar replication fork.
Topics: Animals; DNA Primase; DNA Helicases; DNA-Directed DNA Polymerase; Arabidopsis; Mitochondria; Zinc Fingers; Ribonucleotides; DNA Replication; Bacteriophage T7
PubMed: 37803262
DOI: 10.1186/s12870-023-04477-4 -
Nucleic Acids Research Apr 2024It is well-established that, through canonical functions in transcription and DNA repair, the tumor suppressor p53 plays a central role in safeguarding cells from the...
It is well-established that, through canonical functions in transcription and DNA repair, the tumor suppressor p53 plays a central role in safeguarding cells from the consequences of DNA damage. Recent data retrieved in tumor and stem cells demonstrated that p53 also carries out non-canonical functions when interacting with the translesion synthesis (TLS) polymerase iota (POLι) at DNA replication forks. This protein complex triggers a DNA damage tolerance (DDT) mechanism controlling the DNA replication rate. Given that the levels of p53 trigger non-binary rheostat-like functions in response to stress or during differentiation, we explore the relevance of the p53 levels for its DDT functions at the fork. We show that subtle changes in p53 levels modulate the contribution of some DDT factors including POLι, POLη, POLζ, REV1, PCNA, PRIMPOL, HLTF and ZRANB3 to the DNA replication rate. Our results suggest that the levels of p53 are central to coordinate the balance between DDT pathways including (i) fork-deceleration by the ZRANB3-mediated fork reversal factor, (ii) POLι-p53-mediated fork-slowing, (iii) POLι- and POLη-mediated TLS and (iv) PRIMPOL-mediated fork-acceleration. Collectively, our study reveals the relevance of p53 protein levels for the DDT pathway choice in replicating cells.
Topics: Tumor Suppressor Protein p53; DNA-Directed DNA Polymerase; Humans; DNA Damage; DNA Replication; DNA Polymerase iota; Proliferating Cell Nuclear Antigen; DNA Repair; Nucleotidyltransferases; DNA-Binding Proteins; Multifunctional Enzymes; DNA Primase; DNA Damage Tolerance
PubMed: 38321962
DOI: 10.1093/nar/gkae061 -
BioRxiv : the Preprint Server For... Sep 2023The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity...
The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of polα-primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5'-end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer/template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα-primase.
PubMed: 36993335
DOI: 10.1101/2023.03.16.533013 -
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