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BioRxiv : the Preprint Server For... Jan 2024Telomerase adds G-rich telomeric repeats to the 3' ends of telomeres, counteracting telomere shortening caused by loss of telomeric 3' overhangs during leading-strand...
Telomerase adds G-rich telomeric repeats to the 3' ends of telomeres, counteracting telomere shortening caused by loss of telomeric 3' overhangs during leading-strand DNA synthesis ("the end-replication problem"). We report a second end-replication problem that originates from the incomplete duplication of the C-rich telomeric repeat strand by lagging-strand synthesis. This problem is solved by CST-Polymeraseα(Polα)-primase fill-in synthesis. priming for lagging-strand DNA replication does not occur on the 3' overhang and lagging-strand synthesis stops in an ~150-nt zone more than 26 nt from the end of the template. Consistent with the data, lagging-end telomeres of cells lacking CST-Polα-primase lost ~50-60 nt of CCCTAA repeats per population doubling (PD). The C-strands of leading-end telomeres shortened by ~100 nt/PD, reflecting the generation of 3' overhangs through resection. The measured overall C-strand shortening in absence of CST-Polα-primase fill-in is consistent with the combined effects of incomplete lagging-strand synthesis and 5' resection at the leading-ends. We conclude that canonical DNA replication creates two telomere end-replication problems that require telomerase to maintain the G-strand and CST-Polα-primase to maintain the C-strand.
PubMed: 37961611
DOI: 10.1101/2023.10.26.564248 -
Nucleic Acids Research Dec 2023Telomeres are nucleoprotein complexes that protect the chromosome-ends from eliciting DNA repair while ensuring their complete duplication. Pot1 is a subunit of telomere...
Telomeres are nucleoprotein complexes that protect the chromosome-ends from eliciting DNA repair while ensuring their complete duplication. Pot1 is a subunit of telomere capping complex that binds to the G-rich overhang and inhibits the activation of DNA damage checkpoints. In this study, we explore new functions of fission yeast Pot1 by using a pot1-1 temperature sensitive mutant. We show that pot1 inactivation impairs telomere DNA replication resulting in the accumulation of ssDNA leading to the complete loss of telomeric DNA. Recruitment of Stn1 to telomeres, an auxiliary factor of DNA lagging strand synthesis, is reduced in pot1-1 mutants and overexpression of Stn1 rescues loss of telomeres and cell viability at restrictive temperature. We propose that Pot1 plays a crucial function in telomere DNA replication by recruiting Stn1-Ten1 and Polα-primase complex to telomeres via Tpz1, thus promoting lagging-strand DNA synthesis at stalled replication forks.
Topics: DNA Replication; DNA-Binding Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Shelterin Complex; Telomere; Telomere-Binding Proteins; Chromosomes, Fungal
PubMed: 37953281
DOI: 10.1093/nar/gkad1036 -
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 -
Molecular and Clinical Oncology Dec 2023Genomic imprinting, an epigenetic mechanism that regulates gene expression from parental chromosomes, holds substantial relevance in multiple cancers, including...
Genomic imprinting, an epigenetic mechanism that regulates gene expression from parental chromosomes, holds substantial relevance in multiple cancers, including hematopoietic malignancies. In the present study, the expression of a panel of 16 human imprinted genes in bone marrow samples from 64 patients newly diagnosed with cytogenetically normal-acute myeloid leukemia (CN-AML) were examined alongside peripheral blood samples from 85 healthy subjects. The validated findings of the present study revealed significant upregulation of seven genes [COPI coat complex subunit gamma 2 (), H19 imprinted maternally expressed transcript (), insulin like growth factor 2 (), PEG3 antisense RNA 1 (), DNA primase subunit 2 (), solute carrier family 22 member 3 and Zinc finger protein 215 ()] in patients with CN-AML (P<0.001). Notably, the expression level of exhibited an inverse association with the survival duration of the patients (P=0.018), establishing it as a predictive marker for two- and five-year survival in patients with CN-AML. Kaplan-Meier analysis demonstrated that patients with lower expression had superior two- and five-year survival rates compared with those with higher expression. The results of the present study highlighted the association between loss of imprinting and leukemogenesis in CN-AML, underscoring the significance of imprinting loss as a prognostic indicator for unfavorable two- and five-year survival in CN-AML patients.
PubMed: 37920417
DOI: 10.3892/mco.2023.2690 -
Viruses Sep 2023Bovine herpesvirus type 1 (BoHV-1) is an important agricultural pathogen that infects cattle and other ruminants worldwide. Though it was first sequenced and annotated...
Bovine herpesvirus type 1 (BoHV-1) is an important agricultural pathogen that infects cattle and other ruminants worldwide. Though it was first sequenced and annotated over twenty years ago, the Cooper strain, used in this study, was sequenced as recently as 2012 and is currently said to encode 72 unique proteins. However, tandem mass spectrometry has identified several peptides produced during active infection that align with the BoHV-1 genome in unannotated regions. One of these abundant peptides, "ORF M", aligned antisense to the DNA helicase/primase protein U5. This study characterizes the novel transcript and its protein product and provides evidence to support the existence of homolog protein-coding genes in other Herpesviruses.
Topics: Animals; Cattle; Herpesvirus 1, Bovine; Base Sequence; Simplexvirus; DNA Primase; Peptides; Herpesviridae Infections
PubMed: 37896756
DOI: 10.3390/v15101977 -
Journal of Molecular Biology Dec 2023DNA replication in eukaryotes relies on the synthesis of a ∼30-nucleotide RNA/DNA primer strand through the dual action of the heterotetrameric polymerase α-primase...
DNA replication in eukaryotes relies on the synthesis of a ∼30-nucleotide RNA/DNA primer strand through the dual action of the heterotetrameric polymerase α-primase (pol-prim) enzyme. Synthesis of the 7-10-nucleotide RNA primer is regulated by the C-terminal domain of the primase regulatory subunit (PRIM2C) and is followed by intramolecular handoff of the primer to pol α for extension by ∼20 nucleotides of DNA. Here, we provide evidence that RNA primer synthesis is governed by a combination of the high affinity and flexible linkage of the PRIM2C domain and the surprisingly low affinity of the primase catalytic domain (PRIM1) for substrate. Using a combination of small angle X-ray scattering and electron microscopy, we found significant variability in the organization of PRIM2C and PRIM1 in the absence and presence of substrate, and that the population of structures with both PRIM2C and PRIM1 in a configuration aligned for synthesis is low. Crosslinking was used to visualize the orientation of PRIM2C and PRIM1 when engaged by substrate as observed by electron microscopy. Microscale thermophoresis was used to measure substrate affinities for a series of pol-prim constructs, which showed that the PRIM1 catalytic domain does not bind the template or emergent RNA-primed templates with appreciable affinity. Together, these findings support a model of RNA primer synthesis in which generation of the nascent RNA strand and handoff of the RNA-primed template from primase to polymerase α is mediated by the high degree of inter-domain flexibility of pol-prim, the ready dissociation of PRIM1 from its substrate, and the much higher affinity of the POLA1cat domain of polymerase α for full-length RNA-primed templates.
Topics: Humans; DNA Primase; DNA Primers; DNA Replication; RNA
PubMed: 37884206
DOI: 10.1016/j.jmb.2023.168330 -
Microbiology Spectrum Dec 2023Eukaryotic DNA replication is a highly regulated process that requires multiple replication enzymes assembled onto DNA replication origins. Due to the complexity of the...
Eukaryotic DNA replication is a highly regulated process that requires multiple replication enzymes assembled onto DNA replication origins. Due to the complexity of the cell's DNA replication machinery, most of what we know about cellular DNA replication has come from the study of viral systems. Herein, we focus our study on the assembly of the Kaposi's sarcoma-associated herpesvirus core replication complex and propose a pairwise protein-protein interaction network of six highly conserved viral core replication proteins. A detailed understanding of the interaction and assembly of the viral core replication proteins may provide opportunities to develop new strategies against viral propagation.
Topics: Herpesvirus 8, Human; Viral Proteins; DNA Replication
PubMed: 37874136
DOI: 10.1128/spectrum.02254-23 -
Antiviral Research Nov 2023Herpes is a contagious life-long infection with persistently high incidence and prevalence, causing significant disease worldwide. Current therapies have efficacy...
Herpes is a contagious life-long infection with persistently high incidence and prevalence, causing significant disease worldwide. Current therapies have efficacy against active HSV infections but no impact on the latent viral reservoir in neurons. Thus, despite treatment, disease recurs from latency and the infectious potential remains unaffected within patients. Here, efficacy of the helicase-primase inhibitor (HPI) IM-250 against chronic neuronal HSV infections utilizing two classic herpes in vivo latency/reactivation animal models (intravaginal guinea pig HSV-2 infection model and ocular mouse HSV-1 infection model) is presented. Intermittent therapy of infected animals with 4-7 cycles of IM-250 during latency silences subsequent recurrences analyzed up to 6 months. In contrast to common experience, our studies show that the latent reservoir is indeed accessible to antiviral therapy altering the latent viral reservoir such that reactivation frequency can be reduced significantly by prior IM-250 treatment. We provide evidence that antiviral treatment during HSV latency can reduce future reactivation from the latent reservoir, supporting a conceptual shift in the antiviral field, and reframing what is achievable with respect to therapy of latent neuronal HSV infections.
Topics: Humans; Animals; Mice; Guinea Pigs; DNA Primase; Virus Latency; Herpes Simplex; Herpesvirus 1, Human; Disease Models, Animal; Antiviral Agents
PubMed: 37858763
DOI: 10.1016/j.antiviral.2023.105733 -
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 -
Biochemistry. Biokhimiia Aug 2023Transmission of genetic information depends on successful completion of DNA replication. Genomic DNA is subjected to damage on a daily basis. DNA lesions create... (Review)
Review
Transmission of genetic information depends on successful completion of DNA replication. Genomic DNA is subjected to damage on a daily basis. DNA lesions create obstacles for DNA polymerases and can lead to the replication blockage, formation of DNA breaks, cell cycle arrest, and apoptosis. Cells have evolutionary adapted to DNA damage by developing mechanisms allowing elimination of lesions prior to DNA replication (DNA repair) and helping to bypass lesions during DNA synthesis (DNA damage tolerance). The second group of mechanisms includes the restart of DNA synthesis at the sites of DNA damage by DNA primase-polymerase PrimPol. Human PrimPol was described in 2013. The properties and functions of this enzyme have been extensively studied in recent years, but very little is known about the regulation of PrimPol and association between the enzyme dysfunction and diseases. In this review, we described the mechanisms of human PrimPol regulation in the context of DNA replication, discussed in detail interactions of PrimPol with other proteins, and proposed possible pathways for the regulation of human PrimPol activity. The article also addresses the association of PrimPol dysfunction with human diseases.
Topics: Humans; DNA Primase; DNA-Directed DNA Polymerase; DNA Replication; DNA; DNA Damage; Multifunctional Enzymes
PubMed: 37758313
DOI: 10.1134/S0006297923080084