-
Biochemistry Jun 2024DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets...
DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets in multiple pathogens, including , which in 2021 caused >1.5 million deaths worldwide. DNA gyrase is a tetrameric (AB) protein formed from two subunit types: gyrase A (GyrA) carries the breakage-reunion active site, whereas gyrase B (GyrB) catalyzes ATP hydrolysis required for energy transduction and DNA translocation. The GyrB ATPase domains dimerize in the presence of ATP to trap the translocated DNA (T-DNA) segment as a first step in strand passage, for which hydrolysis of one of the two ATPs and release of the resulting inorganic phosphate is rate-limiting. Here, dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations of the dimeric 43 kDa N-terminal fragment of GyrB show how events at the ATPase site (dissociation/hydrolysis of bound nucleotides) are propagated through communication pathways to other functionally important regions of the GyrB ATPase domain. Specifically, our simulations identify two distinct pathways that respectively connect the GyrB ATPase site to the corynebacteria-specific C-loop, thought to interact with GyrA prior to DNA capture, and to the C-terminus of the GyrB transduction domain, which in turn contacts the C-terminal GyrB topoisomerase-primase (TOPRIM) domain responsible for interactions with GyrA and the centrally bound G-segment DNA. The connection between the ATPase site and the C-loop of dimeric GyrB is consistent with the unusual properties of DNA gyrase relative to those from other bacterial species.
Topics: Mycobacterium tuberculosis; DNA Gyrase; Molecular Dynamics Simulation; Adenosine Triphosphatases; Protein Domains; Adenosine Triphosphate; Bacterial Proteins; Signal Transduction
PubMed: 38742407
DOI: 10.1021/acs.biochem.4c00161 -
Proceedings of the National Academy of... Apr 2024The ParABS system is crucial for the faithful segregation and inheritance of many bacterial chromosomes and low-copy-number plasmids. However, despite extensive...
The ParABS system is crucial for the faithful segregation and inheritance of many bacterial chromosomes and low-copy-number plasmids. However, despite extensive research, the spatiotemporal dynamics of the ATPase ParA and its connection to the dynamics and positioning of the ParB-coated cargo have remained unclear. In this study, we utilize high-throughput imaging, quantitative data analysis, and computational modeling to explore the in vivo dynamics of ParA and its interaction with ParB-coated plasmids and the nucleoid. As previously observed, we find that F-plasmid ParA undergoes collective migrations ("flips") between cell halves multiple times per cell cycle. We reveal that a constricting nucleoid is required for these migrations and that they are triggered by a plasmid crossing into the cell half with greater ParA. Using simulations, we show that these dynamics can be explained by the combination of nucleoid constriction and cooperative ParA binding to the DNA, in line with the behavior of other ParA proteins. We further show that these ParA flips act to equally partition plasmids between the two lobes of the constricted nucleoid and are therefore important for plasmid stability, especially in fast growth conditions for which the nucleoid constricts early in the cell cycle. Overall, our work identifies a second mode of action of the ParABS system and deepens our understanding of how this important segregation system functions.
Topics: Plasmids; Escherichia coli; Escherichia coli Proteins; Chromosomes, Bacterial; Bacterial Proteins; Adenosine Triphosphatases; Chromosome Segregation; DNA Primase; DNA, Bacterial
PubMed: 38652748
DOI: 10.1073/pnas.2319205121 -
Journal of Basic Microbiology Jan 2024The transcription factor (TF)-mediated regulatory network controlling lincomycin production in Streptomyces lincolnensis is yet to be fully elucidated despite several...
The transcription factor (TF)-mediated regulatory network controlling lincomycin production in Streptomyces lincolnensis is yet to be fully elucidated despite several types of associated TFs having been reported. SLCG_2919, a tetracycline repressor (TetR)-type regulator, was the first TF to be characterized outside the lincomycin biosynthetic cluster to directly suppress the lincomycin biosynthesis in S. lincolnensis. In this study, improved genomic systematic evolution of ligands by exponential enrichment (gSELEX), an in vitro technique, was adopted to capture additional SLCG_2919-targeted sequences harboring the promoter regions of SLCG_6675, SLCG_4123-4124, SLCG_6579, and SLCG_0139-0140. The four DNA fragments were confirmed by electrophoretic mobility shift assays (EMSAs). Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) showed that the corresponding target genes SLCG_6675 (anthranilate synthase), SLCG_0139 (LysR family transcriptional regulator), SLCG_0140 (beta-lactamase), SLCG_6579 (cytochrome P450), SLCG_4123 (bifunctional DNA primase/polymerase), and SLCG_4124 (magnesium or magnesium-dependent protein phosphatase) in ΔSLCGL_2919 were differentially increased by 3.3-, 4.2-, 3.2-, 2.5-, 4.6-, and 2.2-fold relative to those in the parental strain S. lincolnensis LCGL. Furthermore, the individual inactivation of these target genes in LCGL reduced the lincomycin yield to varying degrees. This investigation expands on the known DNA targets of SLCG_2919 to control lincomycin production and lays the foundation for improving industrial lincomycin yields via genetic engineering of this regulatory network.
Topics: Magnesium; Bacterial Proteins; Anti-Bacterial Agents; Lincomycin; Transcription Factors; Tetracycline; DNA; Gene Expression Regulation, Bacterial; Streptomyces
PubMed: 37562983
DOI: 10.1002/jobm.202300203 -
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 -
Nature Communications Jan 2024
PubMed: 38225289
DOI: 10.1038/s41467-024-44904-0 -
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 -
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 -
Nature Communications Feb 2024It has been extensively studied that the gut microbiome provides animals flexibility to adapt to food variability. Yet, how gut phageome responds to diet variation of...
It has been extensively studied that the gut microbiome provides animals flexibility to adapt to food variability. Yet, how gut phageome responds to diet variation of wild animals remains unexplored. Here, we analyze the eco-evolutionary dynamics of gut phageome in six wild gibbons (Hoolock tianxing) by collecting individually-resolved fresh fecal samples and parallel feeding behavior data for 15 consecutive months. Application of complementary viral and microbial metagenomics recovers 39,198 virulent and temperate phage genomes from the feces. Hierarchical cluster analyses show remarkable seasonal diet variations in gibbons. From high-fruit to high-leaf feeding period, the abundances of phage populations are seasonally fluctuated, especially driven by the increased abundance of virulent phages that kill the Lachnospiraceae hosts, and a decreased abundance of temperate phages that piggyback the Bacteroidaceae hosts. Functional profiling reveals an enrichment through horizontal gene transfers of toxin-antitoxin genes on temperate phage genomes in high-leaf season, potentially conferring benefits to their prokaryotic hosts. The phage-host ecological dynamics are driven by the coevolutionary processes which select for tail fiber and DNA primase genes on virulent and temperate phage genomes, respectively. Our results highlight complex phageome-microbiome interactions as a key feature of the gibbon gut microbial ecosystem responding to the seasonal diet.
Topics: Animals; Hylobates; Seasons; Ecosystem; Virome; Diet; Bacteriophages; Fruit; Hylobatidae
PubMed: 38341424
DOI: 10.1038/s41467-024-45663-8 -
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 -
International Journal of Molecular... Apr 2024Polyomavirus (PyV) Large T-antigen (LT) is the major viral regulatory protein that targets numerous cellular pathways for cellular transformation and viral replication....
Polyomavirus (PyV) Large T-antigen (LT) is the major viral regulatory protein that targets numerous cellular pathways for cellular transformation and viral replication. LT directly recruits the cellular replication factors involved in initiation of viral DNA replication through mutual interactions between LT, DNA polymerase alpha-primase (Polprim), and single-stranded DNA binding complex, (RPA). Activities and interactions of these complexes are known to be modulated by post-translational modifications; however, high-sensitivity proteomic analyses of the PTMs and proteins associated have been lacking. High-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) of the immunoprecipitated factors (IPMS) identified 479 novel phosphorylated amino acid residues (PAARs) on the three factors; the function of one has been validated. IPMS revealed 374, 453, and 183 novel proteins associated with the three, respectively. A significant transcription-related process network identified by Gene Ontology (GO) enrichment analysis was unique to LT. Although unidentified by IPMS, the ETS protooncogene 1, transcription factor (ETS1) was significantly overconnected to our dataset indicating its involvement in PyV processes. This result was validated by demonstrating that ETS1 coimmunoprecipitates with LT. Identification of a novel PAAR that regulates PyV replication and LT's association with the protooncogenic Ets1 transcription factor demonstrates the value of these results for studies in PyV biology.
Topics: Phosphorylation; Humans; Proteomics; Virus Replication; DNA Replication; Polyomavirus; Tandem Mass Spectrometry; Proto-Oncogene Protein c-ets-1; Chromatography, Liquid; Antigens, Viral, Tumor; Protein Processing, Post-Translational; DNA, Viral
PubMed: 38674125
DOI: 10.3390/ijms25084540