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Bioorganic & Medicinal Chemistry Letters Jul 2024Helicase-primase is an interesting target for the therapy of herpes simplex virus (HSV) infections. Since amenamevir is already approved for varicella-zoster virus (VZV)...
Helicase-primase is an interesting target for the therapy of herpes simplex virus (HSV) infections. Since amenamevir is already approved for varicella-zoster virus (VZV) and HSV in Japan and pritelivir has received breakthrough therapy status for the treatment of acyclovir-resistant HSV infections in immunocompromised patients, the target has sparked interest in me-too approaches. Here, we describe the attempt to improve nervous tissue penetration in Phaeno Therapeutics drug candidate HN0037 to target the latent reservoir of HSV by installing less polar moieties, mainly a difluorophenyl instead of a pyridyl group, and replacing the primary sulfonamide with a methyl sulfoximine moiety. However, all obtained stereoisomers exhibited a weaker inhibitory activity on HSV-1 and HSV-2.
Topics: Sulfonamides; DNA Primase; Antiviral Agents; Enzyme Inhibitors; Structure-Activity Relationship; DNA Helicases; Herpesvirus 1, Human; Herpesvirus 2, Human; Humans; Molecular Structure; Microbial Sensitivity Tests; Dose-Response Relationship, Drug; Imines
PubMed: 38642810
DOI: 10.1016/j.bmcl.2024.129761 -
Nucleic Acids Research May 2024Protein binding microarrays (PBM), SELEX, RNAcompete and chromatin-immunoprecipitation have been intensively used to determine the specificity of nucleic acid binding...
Protein binding microarrays (PBM), SELEX, RNAcompete and chromatin-immunoprecipitation have been intensively used to determine the specificity of nucleic acid binding proteins. While the specificity of proteins with pronounced sequence specificity is straightforward, the determination of the sequence specificity of proteins of modest sequence specificity is more difficult. In this work, an explorative data analysis workflow for nucleic acid binding data was developed that can be used by scientists that want to analyse their binding data. The workflow is based on a regressor realized in scikit-learn, the major machine learning module for the scripting language Python. The regressor is built on a thermodynamic model of nucleic acid binding and describes the sequence specificity with base- and position-specific energies. The regressor was used to determine the binding specificity of the T7 primase. For this, we reanalysed the binding data of the T7 primase obtained with a custom PBM. The binding specificity of the T7 primase agrees with the priming specificity (5'-GTC) and the template (5'-GGGTC) for the preferentially synthesized tetraribonucleotide primer (5'-pppACCC) but is more relaxed. The dominant contribution of two positions in the motif can be explained by the involvement of the initiating and elongating nucleotides for template binding.
Topics: Bacteriophage T7; Binding Sites; DNA Primase; Protein Array Analysis; Protein Binding; Thermodynamics; Viral Proteins
PubMed: 38597656
DOI: 10.1093/nar/gkae215 -
The FEBS Journal May 2024Several recent cryo-electron microscopy (cryo-EM) studies about the eukaryotic primosome, including the human primosome described by Yin et al. in this issue, have...
Several recent cryo-electron microscopy (cryo-EM) studies about the eukaryotic primosome, including the human primosome described by Yin et al. in this issue, have uncovered the structural intricacies between the RNA primase and the DNA polymerase. These studies show that these two partners tango on DNA to synthesize a hybrid primer composed of ~ 10 nucleotide (nt) RNA and ~ 10-nt DNA. They reveal key intermediate steps involved in this process; from the self-inhibited apo state to the initiation of RNA primer synthesis, RNA primer handover to the polymerase, primer elongation by polymerase, and finally, primer termination and release. Remarkably, the polymerase domain orchestrates all major steps during primer synthesis.
Topics: Humans; Cryoelectron Microscopy; DNA; DNA Polymerase I; DNA Primase; DNA Primers; DNA Replication; RNA
PubMed: 38581152
DOI: 10.1111/febs.17129 -
BioRxiv : the Preprint Server For... Mar 2024The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Polα), initiates DNA synthesis on both chromosome strands by generating chimeric...
The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Polα), initiates DNA synthesis on both chromosome strands by generating chimeric RNA-DNA primers for loading DNA polymerases delta and epsilon (Polε). Replication protein A (RPA) tightly binds to single-stranded DNA strands, protecting them from nucleolytic digestion and unauthorized transactions. We report here that RPA plays a critical role for the human primosome during DNA synthesis across inverted repeats prone to hairpin formation. On other alternatively structured DNA forming a G-quadruplex, RPA provides no assistance for primosome. A stimulatory effect of RPA on DNA synthesis across hairpins was also observed for the catalytic domain of Polα but not of Polε. The important factors for an efficient hairpin bypass by primosome are the high affinity of RPA to DNA based on four DNA-binding domains and the interaction of the winged-helix-turn-helix domain of RPA with Polα. Binding studies indicate that this interaction stabilizes the RPA/Polα complex on the primed template. This work provides insight into a cooperative action of RPA and primosome on DNA, which is critical for DNA synthesis across inverted repeats.
PubMed: 38559116
DOI: 10.1101/2024.03.11.584335 -
Genes Mar 2024The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation... (Review)
Review
The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation events at replication origins, the start of the leading strand synthesis for each replicon, and the numerous initiation events taking place during lagging strand DNA synthesis. In addition, a third mechanism is the re-initiation of DNA synthesis after replication fork stalling, which takes place when DNA lesions hinder the progression of DNA synthesis. The initiation of leading strand synthesis at replication origins is regulated at multiple levels, from the origin recognition to the assembly and activation of replicative helicase, the Cdc45-MCM2-7-GINS (CMG) complex. In addition, the multiple interactions of the CMG complex with the eukaryotic replicative DNA polymerases, DNA polymerase α-primase, DNA polymerase δ and ε, at replication forks play pivotal roles in the mechanism of the initiation reactions of leading and lagging strand DNA synthesis. These interactions are also important for the initiation of signalling at unperturbed and stalled replication forks, "replication stress" events, via ATR (ATM-Rad 3-related protein kinase). These processes are essential for the accurate transfer of the cells' genetic information to their daughters. Thus, failures and dysfunctions in these processes give rise to genome instability causing genetic diseases, including cancer. In their influential review "Hallmarks of Cancer: New Dimensions", Hanahan and Weinberg (2022) therefore call genome instability a fundamental function in the development process of cancer cells. In recent years, the understanding of the initiation processes and mechanisms of human DNA replication has made substantial progress at all levels, which will be discussed in the review.
Topics: Humans; DNA; DNA Replication; DNA Polymerase III; Minichromosome Maintenance Proteins; Genomic Instability
PubMed: 38540419
DOI: 10.3390/genes15030360 -
Journal of the American Chemical Society Apr 2024Primases are crucial enzymes for DNA replication, as they synthesize a short primer required for initiating DNA replication. We herein present time-resolved nuclear...
Primases are crucial enzymes for DNA replication, as they synthesize a short primer required for initiating DNA replication. We herein present time-resolved nuclear magnetic resonance (NMR) spectroscopy in solution and in the solid state to study the initial dinucleotide formation reaction of archaeal pRN1 primase. Our findings show that the helix-bundle domain (HBD) of pRN1 primase prepares the two substrates and then hands them over to the catalytic domain to initiate the reaction. By using nucleotide triphosphate analogues, the reaction is substantially slowed down, allowing us to study the initial dinucleotide formation in real time. We show that the sedimented protein-DNA complex remains active in the solid-state NMR rotor and that time-resolved P-detected cross-polarization experiments allow monitoring the kinetics of dinucleotide formation. The kinetics in the sedimented protein sample are comparable to those determined by solution-state NMR. Protein conformational changes during primer synthesis are observed in time-resolved H-detected experiments at fast magic-angle spinning frequencies (100 kHz). A significant number of spectral changes cluster in the HBD pointing to the importance of the HBD for positioning the nucleotides and the dinucleotide.
Topics: DNA Primase; DNA Replication; Nucleotides; Magnetic Resonance Spectroscopy; Carcinoma, Papillary; Carcinoma, Renal Cell; Thyroid Neoplasms
PubMed: 38538061
DOI: 10.1021/jacs.3c11836 -
ISME Communications Jan 2024Combining multiple displacement amplification (MDA) with metagenomics enables the analysis of samples with extremely low DNA concentrations, making them suitable for...
Combining multiple displacement amplification (MDA) with metagenomics enables the analysis of samples with extremely low DNA concentrations, making them suitable for high-throughput sequencing. Although amplification bias and nonspecific amplification have been reported from MDA-amplified samples, the impact of MDA on metagenomic datasets is not well understood. We compared three MDA methods (i.e. bulk MDA, emulsion MDA, and primase MDA) for metagenomic analysis of two DNA template concentrations (approx. 1 and 100 pg) derived from a microbial community standard "mock community" and two low biomass environmental samples (i.e. borehole fluid and groundwater). We assessed the impact of MDA on metagenome-based community composition, assembly quality, functional profiles, and binning. We found amplification bias against high GC content genomes but relatively low nonspecific amplification such as chimeras, artifacts, or contamination for all MDA methods. We observed MDA-associated representational bias for microbial community profiles, especially for low-input DNA and with the primase MDA method. Nevertheless, similar taxa were represented in MDA-amplified libraries to those of unamplified samples. The MDA libraries were highly fragmented, but similar functional profiles to the unamplified libraries were obtained for bulk MDA and emulsion MDA at higher DNA input and across these MDA libraries for the groundwater sample. Medium to low-quality bins were possible for the high input bulk MDA metagenomes for the most simple microbial communities, borehole fluid, and mock community. Although MDA-based amplification should be avoided, it can still reveal meaningful taxonomic and functional information from samples with extremely low DNA concentration where direct metagenomics is otherwise impossible.
PubMed: 38500705
DOI: 10.1093/ismeco/ycae024 -
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 -
Nature Structural & Molecular Biology May 2024The 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 Xenopus laevis 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.
Topics: DNA Primase; DNA Polymerase I; Animals; Cryoelectron Microscopy; Models, Molecular; Xenopus laevis; DNA Replication; Catalytic Domain; DNA; DNA Primers; RNA; Protein Conformation
PubMed: 38491139
DOI: 10.1038/s41594-024-01227-4 -
Nature May 2024Prokaryotes have evolved intricate innate immune systems against phage infection. Gabija is a highly widespread prokaryotic defence system that consists of two...
Prokaryotes have evolved intricate innate immune systems against phage infection. Gabija is a highly widespread prokaryotic defence system that consists of two components, GajA and GajB. GajA functions as a DNA endonuclease that is inactive in the presence of ATP. Here, to explore how the Gabija system is activated for anti-phage defence, we report its cryo-electron microscopy structures in five states, including apo GajA, GajA in complex with DNA, GajA bound by ATP, apo GajA-GajB, and GajA-GajB in complex with ATP and Mg. GajA is a rhombus-shaped tetramer with its ATPase domain clustered at the centre and the topoisomerase-primase (Toprim) domain located peripherally. ATP binding at the ATPase domain stabilizes the insertion region within the ATPase domain, keeping the Toprim domain in a closed state. Upon ATP depletion by phages, the Toprim domain opens to bind and cleave the DNA substrate. GajB, which docks on GajA, is activated by the cleaved DNA, ultimately leading to prokaryotic cell death. Our study presents a mechanistic landscape of Gabija activation.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Apoproteins; Bacterial Proteins; Bacteriophages; Cryoelectron Microscopy; DNA; DNA Cleavage; Magnesium; Models, Molecular; Protein Binding; Protein Domains; Immunity, Innate; Microbial Viability; Bacillus cereus; Protein Structure, Quaternary; DNA Primase; DNA Topoisomerases
PubMed: 38471529
DOI: 10.1038/s41586-024-07270-x