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BioMed Research International 2020DNA Primase Subunit 1 (PRIM1) is crucial for cancer development and progression. However, there remains a lack of comprehension concerning the clinical implication of...
DNA Primase Subunit 1 (PRIM1) is crucial for cancer development and progression. However, there remains a lack of comprehension concerning the clinical implication of PRIM1 in HCC. Here, aberrant expression of PRIM1 was identified in HCC according to available databases. The prognostic value of PRIM1 in patients presenting with HCC was further assessed based on TCGA data. Gene set enrichment analysis (GSEA) was subsequently conducted to investigate the potential function of PRIM1. Additionally, the correlations between tumor-infiltrating immune cells (TIICs) and PRIM1 expression were evaluated. The data from TCGA, GEO, ONCOMINE, and HCCDB databases illustrated that PRIM1 was overexpressed in HCC tissues, compared to normal liver tissues (all < 0.05). Kaplan-Meier analysis revealed that high PRIM1 expression in HCC was closely correlated with worse overall survival ( < 0.05). The univariate and multivariate analyses illustrated that PRIM1 expression was an independent novel prognostic indicator in HCC. Additionally, the area under the receiver operating characteristic (AUROC) curve for PRIM1 reached 0.8651, indicating the diagnostic significance of PRIM1 in patients with HCC. GSEA showed that PRIM1 overexpression was significantly enriched in several tumor-related signaling pathways. Besides, TIIC analysis clarified the association between PRIM1 expression and TIICs in HCC. The findings disclose that PRIM1 profoundly implicated in promoting tumorigenesis might work as a desirable biomarker for HCC.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Hepatocellular; DNA Primase; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Lymphocytes, Tumor-Infiltrating; Male; Middle Aged; Prognosis; RNA, Messenger; Signal Transduction; Young Adult
PubMed: 32908930
DOI: 10.1155/2020/9689312 -
Viruses Aug 2021Acyclovir, valacyclovir, and famciclovir are used for the treatment of herpes simplex virus (HSV) and varicella-zoster virus (VZV) infections. Helicase-primase... (Review)
Review
Acyclovir, valacyclovir, and famciclovir are used for the treatment of herpes simplex virus (HSV) and varicella-zoster virus (VZV) infections. Helicase-primase inhibitors (HPIs) inhibit replication fork progression that separates double DNA strands into two single strands during DNA synthesis. The HPIs amenamevir and pritelivir have novel mechanisms of anti-herpetic action, and their once-daily administration has clinical efficacy for genital herpes. Among HPIs, amenamevir has anti-VZV activity. The concentrations of HSV-1 and VZV required for the 50% plaque reduction of amenamevir were 0.036 and 0.047 μM, respectively. We characterized the features of amenamevir regarding its mechanism, resistance, and synergism with acyclovir. Its antiviral activity was not influenced by the viral replication cycle, in contrast to acyclovir. A clinical trial of amenamevir for herpes zoster demonstrated its non-inferiority to valacyclovir. To date, amenamevir has been successfully used in over 1,240,000 patients with herpes zoster in Japan. Post-marketing surveillance of amenamevir in Japan reported side effects with significant potential risk identified by the Japanese Risk Management Plan, including thrombocytopenia, gingival bleeding, and palpitations, although none of these were serious. The clinical efficacy and safety profiles of amenamevir were established in patients with herpes zoster. Therefore, amenamevir as an HPI opens a new era of anti-herpes therapy.
Topics: Animals; Antiviral Agents; Clinical Trials as Topic; DNA Helicases; DNA Primase; Herpes Genitalis; Herpes Zoster; Herpesvirus 1, Human; Humans; Mice; Oxadiazoles
PubMed: 34452412
DOI: 10.3390/v13081547 -
Nature Feb 2021Break-induced replication (BIR) repairs one-ended double-strand breaks in DNA similar to those formed by replication collapse or telomere erosion, and it has been...
Break-induced replication (BIR) repairs one-ended double-strand breaks in DNA similar to those formed by replication collapse or telomere erosion, and it has been implicated in the initiation of genome instability in cancer and other human diseases. Previous studies have defined the enzymes that are required for BIR; however, understanding of initial and extended BIR synthesis, and of how the migrating D-loop proceeds through known replication roadblocks, has been precluded by technical limitations. Here we use a newly developed assay to show that BIR synthesis initiates soon after strand invasion and proceeds more slowly than S-phase replication. Without primase, leading strand synthesis is initiated efficiently, but is unable to proceed beyond 30 kilobases, suggesting that primase is needed for stabilization of the nascent leading strand. DNA synthesis can initiate in the absence of Pif1 or Pol32, but does not proceed efficiently. Interstitial telomeric DNA disrupts and terminates BIR progression, and BIR initiation is suppressed by transcription proportionally to the transcription level. Collisions between BIR and transcription lead to mutagenesis and chromosome rearrangements at levels that exceed instabilities induced by transcription during normal replication. Together, these results provide fundamental insights into the mechanism of BIR and how BIR contributes to genome instability.
Topics: Chromosomes, Fungal; DNA Breaks, Double-Stranded; DNA Helicases; DNA Primase; DNA Repair; DNA Replication; DNA, Fungal; DNA-Directed DNA Polymerase; Genomic Instability; Kinetics; Mutagenesis; Mutation; S Phase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Telomere; Time Factors; Transcription, Genetic
PubMed: 33473214
DOI: 10.1038/s41586-020-03172-w -
Plants (Basel, Switzerland) Nov 2019Plants are sessile organisms, and their DNA is particularly exposed to damaging agents. The integrity of plant mitochondrial and plastid genomes is necessary for cell... (Review)
Review
Plants are sessile organisms, and their DNA is particularly exposed to damaging agents. The integrity of plant mitochondrial and plastid genomes is necessary for cell survival. During evolution, plants have evolved mechanisms to replicate their mitochondrial genomes while minimizing the effects of DNA damaging agents. The recombinogenic character of plant mitochondrial DNA, absence of defined origins of replication, and its linear structure suggest that mitochondrial DNA replication is achieved by a recombination-dependent replication mechanism. Here, I review the mitochondrial proteins possibly involved in mitochondrial DNA replication from a structural point of view. A revision of these proteins supports the idea that mitochondrial DNA replication could be replicated by several processes. The analysis indicates that DNA replication in plant mitochondria could be achieved by a recombination-dependent replication mechanism, but also by a replisome in which primers are synthesized by three different enzymes: Mitochondrial RNA polymerase, Primase-Helicase, and Primase-Polymerase. The recombination-dependent replication model and primers synthesized by the Primase-Polymerase may be responsible for the presence of genomic rearrangements in plant mitochondria.
PubMed: 31766564
DOI: 10.3390/plants8120533 -
Nature May 2022During the initiation of DNA replication, oligonucleotide primers are synthesized de novo by primases and are subsequently extended by replicative polymerases to...
During the initiation of DNA replication, oligonucleotide primers are synthesized de novo by primases and are subsequently extended by replicative polymerases to complete genome duplication. The primase-polymerase (Prim-Pol) superfamily is a diverse grouping of primases, which includes replicative primases and CRISPR-associated primase-polymerases (CAPPs) involved in adaptive immunity. Although much is known about the activities of these enzymes, the precise mechanism used by primases to initiate primer synthesis has not been elucidated. Here we identify the molecular bases for the initiation of primer synthesis by CAPP and show that this mechanism is also conserved in replicative primases. The crystal structure of a primer initiation complex reveals how the incoming nucleotides are positioned within the active site, adjacent to metal cofactors and paired to the templating single-stranded DNA strand, before synthesis of the first phosphodiester bond. Furthermore, the structure of a Prim-Pol complex with double-stranded DNA shows how the enzyme subsequently extends primers in a processive polymerase mode. The structural and mechanistic studies presented here establish how Prim-Pol proteins instigate primer synthesis, revealing the requisite molecular determinants for primer synthesis within the catalytic domain. This work also establishes that the catalytic domain of Prim-Pol enzymes, including replicative primases, is sufficient to catalyse primer formation.
Topics: Catalytic Domain; DNA; DNA Primase; DNA Primers; DNA Replication
PubMed: 35508653
DOI: 10.1038/s41586-022-04695-0 -
The Journal of Biological Chemistry Jun 2022The T7 primase-helicase plays a pivotal role in the replication of T7 DNA. Using affinity isolation of peptide-nucleic acid crosslinks and mass spectrometry, we identify...
The T7 primase-helicase plays a pivotal role in the replication of T7 DNA. Using affinity isolation of peptide-nucleic acid crosslinks and mass spectrometry, we identify protein regions in the primase-helicase and T7 DNA polymerase that form contacts with the RNA primer and DNA template. The contacts between nucleic acids and the primase domain of the primase-helicase are centered in the RNA polymerase subdomain of the primase domain, in a cleft between the N-terminal subdomain and the topoisomerase-primase fold. We demonstrate that residues along a beta sheet in the N-terminal subdomain that contacts the RNA primer are essential for phage growth and primase activity in vitro. Surprisingly, we found mutations in the primase domain that had a dramatic effect on the helicase. Substitution of a residue conserved in other DnaG-like enzymes, R84A, abrogates both primase and helicase enzymatic activities of the T7 primase-helicase. Alterations in this residue also decrease binding of the primase-helicase to ssDNA. However, mass photometry measurements show that these mutations do not interfere with the ability of the protein to form the active hexamer.
Topics: Amino Acid Sequence; Bacteriophage T7; DNA; DNA Helicases; DNA Primase; Mutation; Viral Proteins
PubMed: 35500649
DOI: 10.1016/j.jbc.2022.101996 -
Antimicrobial Agents and Chemotherapy Sep 2021The antiherpetic drug amenamevir (AMNV) inhibits the helicase-primase complex of herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus directly as well as...
The antiherpetic drug amenamevir (AMNV) inhibits the helicase-primase complex of herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus directly as well as inhibiting the replication of these viruses. Although several mutated HSV viruses resistant to helicase-primase inhibitors have been reported, the mutations contributing to the resistance remain unclear, as recombinant viruses containing a single mutation have not been analyzed. We obtained AMNV-resistant viruses with amino acid substitutions by several passages under AMNV treatment. Twenty HSV-1 and 19 HSV-2 mutants with mutation(s) in UL5 helicase and/or UL52 primase, but not in cofactor UL8, were isolated. The mutations in UL5 were located downstream of motif IV, with UL5 K356N in HSV-1 and K355N in HSV-2, in particular, identified as having the highest frequency, which was 9/20 and 9/19, respectively. We generated recombinant AMNV-resistant HSV-1 with a single amino acid substitution using bacterial artificial chromosome (BAC) mutagenesis. As a result, G352C in UL5 helicase and F360C/V and N902T in UL52 primase were identified as novel mutations. The virus with K356N in UL5 showed 10-fold higher AMNV resistance than did other mutants and showed equivalent viral growth and virulence as the parent HSV-1, although other mutants showed attenuated virulence. All recombinant viruses were susceptible to the other antiherpetic drugs, acyclovir and foscarnet. In conclusion, based on BAC mutagenesis, this study identified, for the first time, mutations in UL5 and UL52 that contributed to AMNV resistance and found that a mutant with the most frequent K356N mutation in HSV-1 maintained viral growth and virulence equivalent to the parent virus.
Topics: DNA Helicases; DNA Primase; Herpesvirus 1, Human; Oxadiazoles; Viral Proteins
PubMed: 34228537
DOI: 10.1128/AAC.00494-21 -
Nature Communications Aug 2021Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that...
Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase α (Pol α)-primase in tandem duplication formation at breaks having complementary 3' ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3' tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3' overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells.
Topics: Animals; Base Sequence; Cell Cycle Proteins; Cell Line; Cells, Cultured; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Polymerase I; DNA Primase; DNA, Single-Stranded; DNA-Binding Proteins; Humans; Mice; Microsatellite Repeats; Multiprotein Complexes; Mutation; Telomere; Telomere-Binding Proteins; Tumor Suppressor p53-Binding Protein 1
PubMed: 34376693
DOI: 10.1038/s41467-021-25154-w -
PloS One 2019The spatial organization of DNA is mediated by the Par protein system in some bacteria. ParB binds specifically to the parS sequence on DNA and orchestrates its motion...
The spatial organization of DNA is mediated by the Par protein system in some bacteria. ParB binds specifically to the parS sequence on DNA and orchestrates its motion by interacting with ParA bound to the nucleoid. In the case of plasmids, a single ParB bound plasmid is observed to execute oscillations between cell poles while multiple plasmids eventually settle at equal distances from each other along the cell's length. While the potential mechanism underlying the ParA-ParB interaction has been discussed, it remains unclear whether ParB-complex oscillations are stable limit cycles or merely decaying transients to a fixed point. How are dynamics affected by substrate length and the number of complexes? We present a deterministic model for ParA-ParB driven DNA segregation where the transition between stable arrangements and oscillatory behaviour depends only on five parameters: ParB-complex number, substrate length, ParA concentration, ParA hydrolysis rate and the ratio of the lengthscale over which the ParB complex stimulates ParA hydrolysis to the lengthscale over which ParA interacts with the ParB complex. When the system is buffered and the ParA rebinding rate is constant we find that ParB-complex dynamics is independent of substrate length and complex number above a minimum system size. Conversely, when ParA resources are limited, we find that changing substrate length and increasing complex number leads to counteracting mechanisms that can both generate or subdue oscillatory dynamics. We argue that cells may be poised near a critical level of ParA so that they can transition from oscillatory to fixed point dynamics as the cell cycle progresses so that they can both measure their size and faithfully partition their genetic material. Lastly, we show that by modifying the availability of ParA or depletion zone size, we can capture some of the observed differences in ParB-complex positioning between replicating chromosomes in B. subtilis cells and low-copy plasmids in E. coli cells.
Topics: Bacillus subtilis; Cell Cycle; DNA Primase; DNA Replication; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins
PubMed: 31318889
DOI: 10.1371/journal.pone.0218520 -
Chemical Science Jul 2020Growing experimental evidence indicates that iron-sulfur proteins play key roles in DNA repair and replication. In particular, charge transport between [FeS] clusters,...
Growing experimental evidence indicates that iron-sulfur proteins play key roles in DNA repair and replication. In particular, charge transport between [FeS] clusters, mediated by proteins and DNA, may convey signals to coordinate enzyme action. Human primase is a well studied [FeS] protein, and its p58c domain (which contains an [FeS] cluster) plays a role in the initiation of DNA replication. The Y345C mutation in p58c is linked to gastric tumors and may influence the protein-mediated charge transport. The complexity of protein-DNA systems, and the intricate electronic structure of [FeS] clusters, have impeded progress into understanding functional charge transport in these systems. In this study, we built force fields to describe the high potential [FeS] cluster in both oxidation states. The parameterization is compatible with AMBER force fields and enabled well-balanced molecular dynamics simulations of the p58c-RNA/DNA complex relevant to the initiation of DNA replication. Using the molecular mechanics Poisson-Boltzmann and surface area solvation method on the molecular dynamics trajectories, we find that the p58c mutation induces a modest change in the p58c-duplex binding free energy in agreement with recent experiments. Through kinetic modeling and analysis, we identify key features of the main charge transport pathways in p58c. In particular, we find that the Y345C mutation partially changes the composition and frequency of the most efficient (and potentially relevant to the biological function) charge transport pathways between the [FeS] cluster and the duplex. Moreover, our approach sets the stage for a deeper understanding of functional charge transfer in [FeS] protein-DNA complexes.
PubMed: 33250976
DOI: 10.1039/d0sc02245d