-
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 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 -
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 -
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 -
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