DNA Replication Enzymes Research Articles

Revolutionizing Quinolone Development for DNA Gyrase Targeting; Discovering the Promising Approach to Fighting Microbial Infections

Abstract: DNA gyrase is a type II topoisomerase enzyme that can cause negative supercoiling in DNA by using the energy produced by ATP hydrolysis. There are two main types of topoi-somerases: type I and type II. Type I enzymes cut a single strand of DNA and are further clas-sified as type IA if they connect to a 5′ phosphate of DNA, or type IB if they link to a 3′ phos-phate. Type II topoisomerases break both strands, creating a staggered double-strand break. Antimicrobial resistance is a major concern for the global healthcare system. Resistance is the ability of microorganisms to neutralize and withstand antimicrobial drugs previously used to treat microbial infections. Some known classes of DNA gyrase inhibitors are coumarins, cyclo-thialidines, and quinolones. Antimicrobial medicines such as quinolones have been widely used to treat microbiological diseases. However, the increased use of quinolones has led to the emer-gence of quinolone-resistant bacteria, which poses a serious risk to public health. Microorgan-isms can cause resistance due to changes in the target enzymes, DNA gyrase, and topoisomerase IV, which are responsible for transcription and DNA replication. Additionally, differences in drug entry and efflux may also play a role in resistance. Plasmids that produce the Qnr protein can mediate resistance to quinolones by protecting the quinolone targets from inhibition. This review aims to revolutionize the discovery of quinolone-based antibiotics, specifically targeting DNA gyrase, a critical enzyme in bacterial DNA replication, to enhance the efficacy and spec-ificity of anti-microbail agents against microbial infections.

Antimicrobial Activity of Arthrospira platensis-Mediated Gold Nanoparticles against Streptococcus pneumoniae: A Metabolomic and Docking Study.

The emergence of antibiotic-resistant Streptococcus pneumoniae necessitates the discovery of novel therapeutic agents. This study investigated the antimicrobial potential of green-synthesized gold nanoparticles (AuNPs) fabricated using Arthrospira platensis extract. Characterization using Fourier transform infrared spectroscopy revealed the presence of functional groups such as ketones, aldehydes, and carboxylic acids in the capping agents, suggesting their role in AuNP stabilization. Transmission electron microscopy demonstrated the formation of rod-shaped AuNPs with a mean diameter of 134.8 nm, as determined by dynamic light scattering, and a zeta potential of -27.2 mV, indicating good colloidal stability. The synthesized AuNPs exhibited potent antibacterial activity against S. pneumoniae, with a minimum inhibitory concentration (MIC) of 12 μg/mL, surpassing the efficacy of the control antibiotic, tigecycline. To elucidate the underlying mechanisms of action, an untargeted metabolomic analysis of the A. platensis extract was performed, identifying 26 potential bioactive compounds belonging to diverse chemical classes. In silico studies focused on molecular docking simulations revealed that compound 22 exhibited a strong binding affinity to S. pneumoniae topoisomerase IV, a critical enzyme for bacterial DNA replication. Molecular dynamics simulations further validated the stability of this protein-ligand complex. These findings collectively highlight the promising antimicrobial potential of A. platensis-derived AuNPs and their constituent compounds, warranting further investigation for the development of novel anti-pneumococcal therapeutics.

Ozenoxacin: A Review of Clinical Efficacy on Impetigo Treatment

Impetigo, a common bacterial skin infection primarily affecting children, presents a global prevalence of 11.2%, with higher rates among children aged 2-5 years. Caused mainly by Staphylococcus aureus and Streptococcus pyogenes, impetigo lesions typically appear on the face, neck, and hands, spreading easily through scratching and close contact. Effective disease control is essential to alleviate symptoms, prevent complications such as rheumatic heart disease, and reduce transmission rates. Treatment with antimicrobial agents, especially topical antibiotics, is recommended for localized impetigo. Oral antibiotics are reserved for extensive or systemic infections and outbreaks. However, the emergence of antibiotic-resistant strains, including methicillin-resistant S. aureus (MRSA), poses a significant challenge. There is a pressing need for alternative antimicrobial agents effective against resistant strains. Ozenoxacin, a new topical quinolone, inhibits DNA gyrase and topoisomerase IV, essential enzymes for bacterial DNA replication. Unlike other quinolones, ozenoxacin effectively inhibits both enzymes at low concentrations, penetrating bacterial cells rapidly and achieving high intrabacterial concentrations. In tests against S. aureus and S. pyogenes, ozenoxacin exhibited superior bactericidal activity compared to mupirocin and fusidic acid, achieving a 3-log reduction in colony-forming units within 4 hours. Clinical studies have demonstrated the efficacy and safety of ozenoxacin 1% cream in treating impetigo, with placebo-controlled trials confirming its therapeutic effectiveness. Ozenoxacin holds promise as a valuable addition to the armamentarium against impetigo, particularly in the face of rising antibiotic resistance.impetigo, therapy, ozenoxacin

Initial Primer Synthesis of a DNA Primase Monitored by Real-Time NMR Spectroscopy.

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 31P-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 1H-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.

Non-canonical amino acids uncover the significant impact of Tyr671 on Taq DNA polymerase catalytic activity.

Responsible for synthesizing the complementary strand of the DNA template, DNA polymerase is a crucial enzyme in DNA replication, recombination and repair. A highly conserved tyrosine (Tyr), located at the C-terminus of the O-helix in family A DNA polymerases, plays a critical role in enzyme activity and fidelity. Here, we combined the technology of genetic code extension to incorporate non-canonical amino acids and molecular dynamics (MD) simulations to uncover the mechanisms by which Tyr671 impacts substrate binding and conformation transitions in a DNA polymerase from Thermus aquaticus. Five non-canonical amino acids, namely l-3,4-dihydroxyphenylalanine (l-DOPA), p-aminophenylalanine (pAF), p-acetylphenylalanine (pAcF), p-cyanophenylalanine (pCNF) and p-nitrophenylalanine (pNTF), were individually incorporated at position 671. Strikingly, Y671pAF and Y671DOPA were active, but with lower activity compared to Y671F and wild-type. Y671pAF showed a higher fidelity than the Y671F, despite both possessing lower fidelity than the wild-type. Metadynamics and long-timescale MD simulations were carried out to probethe role of mutations in affecting protein structure, including open conformation, open-to-closed conformation transition, closed conformation, and closed-to-open conformation transition. The MD simulations clearly revealed that the size of the 671 amino acid residue and interactions with substrate or nearby residues were critical for Tyr671 to determine enzyme activity and fidelity.

Disenfranchised DNA: biochemical analysis of mutant øX174 DNA-binding proteins may further elucidate the evolutionary significance of the unessential packaging protein A.

Most icosahedral DNA viruses package and condense their genomes into pre-formed, volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging are specific to single-stranded (ss) DNA micro- and parvoviruses. Before packaging, ~120 copies of the øX174 DNA-binding protein J interact with double-stranded DNA. 60 J proteins enter the procapsid with the ssDNA genome, guiding it between 60 icosahedrally ordered DNA-binding pockets formed by the capsid proteins. Although J proteins are small, 28-37 residues in length, they have two domains. The basic, positively charged N-terminus guides the genome between binding pockets, whereas the C-terminus acts as an anchor to the capsid's inner surface. Three C-terminal aromatic residues, W30, Y31, and F37, interact most extensively with the coat protein. Their corresponding codons were mutated, and the resulting strains were biochemically and genetically characterized. Depending on the mutation, the substitutions produced unstable packaging complexes, unstable virions, infectious progeny, or particles packaged with smaller genomes, the latter being a novel phenomenon. The smaller genomes contained internal deletions. The juncture sequences suggest that the unessential A* (A star) protein mediates deletion formation.IMPORTANCEUnessential but strongly conserved gene products are understudied, especially when mutations do not confer discernable phenotypes or the protein's contribution to fitness is too small to reliably determine in laboratory-based assays. Consequently, their functions and evolutionary impact remain obscure. The data presented herein suggest that microvirus A* proteins, discovered over 40 years ago, may hasten the termination of non-productive packaging events. Thus, performing a salvage function by liberating the reusable components of the failed packaging complexes, such as DNA templates and replication enzymes.

Striking efficacy of a vaccine targeting TOP2A for triple-negative breast cancer immunoprevention

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that has a poor prognosis. TOP2A is a key enzyme in DNA replication and is a therapeutic target for breast and other cancers. TOP2A-specific Th1-promoting epitopes with optimal binding affinity to MHC II were identified using a combined scoring system. The multi-peptide TOP2A vaccine elicited a robust immunologic response in immunized mice, as demonstrated by the significant production of Th1 cytokines from immunized animals’ splenocytes stimulated in vitro with TOP2A peptides. Anti-tumor efficacy of the TOP2A vaccine was demonstrated in a syngeneic TNBC mouse model, in which pre-graft preventive vaccination was associated with significantly decreased tumor growth as compared to adjuvant control. In a genetically engineered mouse (GEM) model of TNBC, vaccinated animals demonstrated a significant reduction in tumor incidence and average tumor volume compared to adjuvant control. Finally, we examined TCR sequences in CD4 tumor Infiltrating lymphocytes (TIL) from vaccinated mice and found that the TIL contained TCR sequences specific to the three vaccine peptides. These data indicate that our newly developed multi-peptide TOP2A vaccine is highly immunogenic, elicits TILs with vaccine specific TCRs, and is highly effective in preventing and intercepting TNBC development and progression in vivo.

Inhibition of Topoisomerases by Metal Thiosemicarbazone Complexes.

Topoisomerases, common targets for anti-cancer therapeutics, are crucial enzymes for DNA replication, transcription, and many other aspects of DNA metabolism. The potential anti-cancer effects of thiosemicarbazones (TSC) and metal-TSC complexes have been demonstrated to target several biological processes, including DNA metabolism. Human topoisomerases were discovered among the molecular targets for TSCs, and metal-chelated TSCs specifically displayed significant inhibition of topoisomerase II. The processes by which metal-TSCs or TSCs inhibit topoisomerases are still being studied. In this brief review, we summarize the TSCs and metal-TSCs that inhibit various types of human topoisomerases, and we note some of the key unanswered questions regarding this interesting class of diverse compounds.

Abstract 1636: Generation of novel potent human TREX1 inhibitors facilitated by crystallography

Abstract Background: STING is an innate immune sensor critical for the development of immunity. Tumor cells can inactivate the STING pathway to avoid immune recognition, emphasizing its importance in generating tumor-specific immunity. Broad activation of STING in advanced cancers may be required to initiate CD8+ T cell priming against unique antigenic repertoires among distinct metastases and to reverse an immune-suppressive TME. Due its ubiquitous expression, systemic delivery of untargeted STING agonists may not achieve a therapeutic index. In contrast, expression of the cytosolic DNAse 3’ repair exonuclease (TREX1) is upregulated in tumor cells in response to genomic instability, inflammatory stimuli, and DNA replication, providing an opportunity for selective activation of the STING pathway. In addition to modulating cGAS/STING signaling, interaction with DNA replication enzymes that generate immunogenic DNA waste highlight a facet of TREX1 biology that may inform clinical development of targeted inhibitors. Methods: Using a structure-based drug design strategy, we designed and optimized small-molecule inhibitors of TREX1 with drug-like physicochemical properties that were profiled in biochemical and cell-based assays. X-ray crystallography studies, thermal shift, and biochemical assays were employed to determine mechanism of action. We evaluated the in vivo profile of select compounds. Results: Inhibitors of TREX1 with < 100 µM potency were optimized into a series with nanomolar potency against purified, recombinant murine and human TREX1 protein in biochemical assays. Inhibitors had similar IC50 values against TREX1 nuclease in an intact cell-based assay. Finally, TSA results demonstrated that compound interaction required magnesium. To our knowledge, we produced the first high-resolution co-crystal structures of inhibitor-bound human TREX1. We used this to dissect mouse- and human-specific interactions, confirming MOA predicted by earlier modeling efforts. Lead compounds demonstrated good bioavailability and achieved exposures necessary for target engagement in mouse models and resulted in tumor growth inhibition when combined with sub-therapeutic doses of doxorubicin. Conclusions: We present the identification and characterization of a potential first-in-class TREX1 inhibitor with nanomolar potency against human and mouse TREX1. Treatment with TREX1 inhibitors conferred profound anti-tumor activity when combined with DNA-damaging agents. Here we demonstrate that targeting TREX1 can specifically and locally engage the STING pathway in the tumor microenvironment, enhance tumor-specific immunity, and provide therapeutic benefit. Citation Format: Valerie Chen, Brian Francica, David Hsieh, Dave Freund, Anja Holtz, Saheli Samanta, Jamie Cope, Ryan Clark, Peppi Prasit, Anne Moon, Henry Johnson, Thomas Dubensky, Dara Burdette. Generation of novel potent human TREX1 inhibitors facilitated by crystallography [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1636.

The interplay between noncoding RNAs and p21 signaling in gastrointestinal cancer: from tumorigenesis to metastasis.

Non-coding RNAs (ncRNAs) are emerging as important regulators in various pathological conditions, including human cancers. NcRNAs exert potentially crucial effects on cell cycle progression, proliferation, and invasion in cancer cells by targeting various cell cycle-related proteins at transcriptional and post-transcriptional levels. As one of the key cell cycle regulatory proteins, p21 is involved in various processes, including the cellular response to DNA damage, cell growth, invasion, metastasis, apoptosis, and senescence. P21 has been shown to have either a tumor-suppressive or oncogenic effect depending on the cellular localization and posttranslational modifications. P21 exerts a significant regulatory effect on both G1/S and G2/M checkpoints by regulating the function of cyclin-dependent kinase enzymes (CDKs) or interacting with proliferating cell nuclear antigen (PCNA). P21 has an important effect on the cellular response to DNA damage by separating DNA replication enzymes from PCNA and inhibiting DNA synthesis resulting in G1 phase arrest. Furthermore, p21 has been shown to negatively regulate the G2/M checkpoint through the inactivation of cyclin-CDK complexes. In response to any cell damage caused by genotoxic agents, p21 exerts its regulatory effects by nuclear preservation of cyclin B1-CDK1 and preventing their activation. Notably, several ncRNAs, including lncRNAs and miRNAs, have been shown to be involved in tumor initiation and progression through the regulation of the p21 signaling axis. In this review, we discuss the miRNA/lncRNA-dependent mechanisms that regulate p21 and their effects on gastrointestinal tumorigenesis. A better understanding of the regulatory effects of ncRNAs on the p21 signaling may help to discover novel therapeutic targets in gastrointestinal cancer.

Indolin-2-one Nitroimidazole Antibiotics Exhibit an Unexpected Dual Mode of Action.

Nitroimidazoles such as metronidazole are used as anti-infective drugs against anaerobic bacteria. Upon in vivo reduction of the nitro group, reactive radicals damage DNA and proteins in the absence of oxygen. Unexpectedly, a recent study of nitroimidazoles linked to an indolin-2-one substituent revealed potent activities against aerobic bacteria. This suggests a different, yet undiscovered mode of action (MoA). To decipher this MoA, we first performed whole proteome analysis of compound-treated cells, revealing an upregulation of bacteriophage-associated proteins, indicative of DNA damage. Since DNA binding of the compound was not observed, we applied activity-based protein profiling (ABPP) for direct target discovery. Labeling studies revealed topoisomerase IV, an essential enzyme for DNA replication, as the most enriched hit in pathogenic Staphylococcus aureus cells. Subsequent topoisomerase assays confirmed the inhibition of DNA decatenation in the presence of indolin-2-one nitroimidazole with an activity comparable to ciprofloxacin, a known inhibitor of this enzyme. Furthermore, we determined significantly increased redox potentials of indolin-2-one nitroimidazoles compared to classic 5-nitroimidazoles such as metronidazole, which facilitates in vivo reduction. Overall, this study unravels that indolin-2-one-functionalized nitroimidazoles feature an unexpected dual MoA: first, the direct inhibition of the topoisomerase IV and second the classic nitroimidazole MoA of reductive bioactivation leading to damaging reactive species. Importantly, this dual MoA impairs resistance development. Given the clinical application of this compound class, the new mechanism could be a starting point to mitigate resistance.

The iron-sulfur cluster is essential for DNA binding by human DNA polymerase ε

DNA polymerase ε (Polε) is a key enzyme for DNA replication in eukaryotes. Recently it was shown that the catalytic domain of yeast Polε (PolεCD) contains a [4Fe-4S] cluster located at the base of the processivity domain (P-domain) and coordinated by four conserved cysteines. In this work, we show that human PolεCD (hPolεCD) expressed in bacterial cells also contains an iron-sulfur cluster. In comparison, recombinant hPolεCD produced in insect cells contains significantly lower level of iron. The iron content of purified hPolECD samples correlates with the level of DNA-binding molecules, which suggests an important role of the iron-sulfur cluster in hPolε interaction with DNA. Indeed, mutation of two conserved cysteines that coordinate the cluster abolished template:primer binding as well as DNA polymerase and proofreading exonuclease activities. We propose that the cluster regulates the conformation of the P-domain, which, like a gatekeeper, controls access to a DNA-binding cleft for a template:primer. The binding studies demonstrated low affinity of hPolεCD to DNA and a strong effect of salt concentration on stability of the hPolεCD/DNA complex. Pre-steady-state kinetic studies have shown a maximal polymerization rate constant of 51.5 s−1 and a relatively low affinity to incoming dNTP with an apparent KD of 105 µM.

The Role of Iron in DNA and Genomic Instability in Cancer, a Target for Iron Chelators That Can Induce ROS

Iron is a key metal involved in several biological processes such as DNA replication and repair, cellular proliferation and cell cycle regulation. Excess volumes of labile iron are toxic and can lead to the production of ROS (reactive oxygen species) via Fenton chemistry. Due to this reactive nature, it can contribute to DNA damage and genomic instability. Therefore, excess iron in the labile iron pool is associated with cancer, which has made the labile iron pool a crucial target for anticancer therapy by targeting iron. This iron can be incorporated into essential enzymes such as ribonucleotide reductase (RnR). Over several decades of research, iron chelators function as more than just RnR inhibitors. Indeed, a plethora of iron chelator mechanisms can result in therapeutic properties that can target critical steps of cancer cells’ aberrant biological abilities such as proliferation, migration and metastasis. One such mechanism is the production of redox-active complexes that can produce toxic levels of ROS in cancer cells. Cancer cells are potentially more susceptible to ROS production or modulation of antioxidant levels. Understanding iron metabolism is vital in targeting cancer. For instance, Fe-S clusters have recently been shown to play crucial roles in cell signalling by ROS through their incorporation into essential DNA replication and repair enzymes. ROS can also degrade Fe-S clusters. Iron chelators that produce toxic levels of ROS, therefore, could also target Fe-S centres. Thus, the design of iron chelators is important, as this can determine if it will participate in redox cycling and produce ROS or if it is solely used to remove iron. This review focuses on alterations in cancer iron metabolism, iron’s role in genomic stability and how the design of chelators can use Fenton chemistry to their advantage to cause DNA damage in cancer cells and potentially inhibit Fe-S centres.

One-step detection of T4 polynucleotide kinase activity based on single particle-confined enzyme reaction and digital particle counting

T4 polynucleotide kinase (T4 PNK) is a pivotal enzyme for DNA replication, recombination, and DNA damage repair. Herein, a robust single particle counting-based assay has been developed for the high-sensitive determination of T4 PNK activity through only a simple one-step reaction. Taking benefit of the exceptional space-confined enzymatic property of T4 PNK towards DNA substrates on a single nanoparticle, the T4 PNK activity can be precisely determined by counting the fluorescence-positive nanoparticles in a digital manner with a total internal reflection fluorescent microscope (TIRFM). Due to the featured spatial-confined enzymatic property of T4 PNK and the single particle counting-based signal readout, T4 PNK can be effectively differentiated from other interfering enzymes. This facile strategy has been also successfully applied to screen T4 PNK inhibitor and accurately determine T4 PNK activity in complex biological samples, paving a potential avenue for the digital analysis of biomarkers.

PrimPol: A Breakthrough among DNA Replication Enzymes and a Potential New Target for Cancer Therapy.

DNA replication can encounter blocking obstacles, leading to replication stress and genome instability. There are several mechanisms for evading this blockade. One mechanism consists of repriming ahead of the obstacles, creating a new starting point; in humans, PrimPol is responsible for carrying out this task. PrimPol is a primase that operates in both the nucleus and mitochondria. In contrast with conventional primases, PrimPol is a DNA primase able to initiate DNA synthesis de novo using deoxynucleotides, discriminating against ribonucleotides. In vitro, PrimPol can act as a DNA primase, elongating primers that PrimPol itself sythesizes, or as translesion synthesis (TLS) DNA polymerase, elongating pre-existing primers across lesions. However, the lack of evidence for PrimPol polymerase activity in vivo suggests that PrimPol only acts as a DNA primase. Here, we provide a comprehensive review of human PrimPol covering its biochemical properties and structure, in vivo function and regulation, and the processes that take place to fill the gap-containing lesion that PrimPol leaves behind. Finally, we explore the available data on human PrimPol expression in different tissues in physiological conditions and its role in cancer.

The [4Fe4S] Cluster of Yeast DNA Polymerase ε Is Redox Active and Can Undergo DNA-Mediated Signaling.

Many DNA replication and DNA repair enzymes have been found to carry [4Fe4S] clusters. The major leading strand polymerase, DNA polymerase ε (Pol ε) from Saccharomyces cerevisiae, was recently reported to have a [4Fe4S] cluster located within the catalytic domain of the largest subunit, Pol2. Here the redox characteristics of the [4Fe4S] cluster in the context of that domain, Pol2CORE, are explored using DNA electrochemistry, and the effects of oxidation and rereduction on polymerase activity are examined. The exonuclease deficient variant D290A/E292A, Pol2COREexo–, was used to limit DNA degradation. While no redox signal is apparent for Pol2COREexo– on DNA-modified electrodes, a large cathodic signal centered at −140 mV vs NHE is observed after bulk oxidation. A double cysteine to serine mutant (C665S/C668S) of Pol2COREexo–, which lacks the [4Fe4S] cluster, shows no similar redox signal upon oxidation. Significantly, protein oxidation yields a sharp decrease in polymerization, while rereduction restores activity almost to the level of untreated enzyme. Moreover, the addition of reduced EndoIII, a bacterial DNA repair enzyme containing [4Fe4S]2+, to oxidized Pol2COREexo– bound to its DNA substrate also significantly restores polymerase activity. In contrast, parallel experiments with EndoIIIY82A, a variant of EndoIII, defective in DNA charge transport (CT), does not show restoration of activity of Pol2COREexo–. We propose a model in which EndoIII bound to the DNA duplex may shuttle electrons through DNA to the DNA-bound oxidized Pol2COREexo– via DNA CT and that this DNA CT signaling offers a means to modulate the redox state and replication by Pol ε.

Responses of Alpha-linolenic acid strain (C-12) from Chlorella sp. L166 to low temperature plasma treatment

LTP treatment was applied to induce a high-content alpha-linolenic acid (ALA) strain (C-12) from Chlorella sp. L166, the ALA content of C-12 was increased by 48.37%. The mechanism of LTP induction were examined. The results showed that LTP could facilitate pH change, induce malondialdehyde (MDA) production, cause protein leakage, and destroyed the microalgae cells. The genes of C-12 encoding pyruvate dehydrogenase (E2) were up-regulated 2.47-fold, and acetyl-CoA carboxylase (ACCase) down-regulated 0.48-fold compared to the wild type, these changed in the direction of ALA accumulation. Furthermore, the enzymes in DNA replication were significantly up regulated. Take ALA and biomass accumulation into account, LTP technology had a positive effect on ALA accumulation. Global view of metabolic variation also suggested that C-12 had an excellent adaptability to the changes of pH and peroxidation of LTP production. C-12 could be a promising ALA source of alternative materials for it do not compete with land.

Replication and partitioning of the apicoplast genome of Toxoplasma gondii is linked to the cell cycle and requires DNA polymerase and gyrase

Apicomplexans are the causative agents of numerous important infectious diseases including malaria and toxoplasmosis. Most of them harbour a chloroplast-like organelle called the apicoplast that is essential for the parasites’ metabolism and survival. While most apicoplast proteins are nuclear encoded, the organelle also maintains its own genome, a 35 kb circle. In this study we used Toxoplasma gondii to identify and characterise essential proteins involved in apicoplast genome replication and to understand how apicoplast genome segregation unfolds over time. We demonstrated that the DNA replication enzymes Prex, DNA gyrase and DNA single stranded binding protein localise to the apicoplast. We show in knockdown experiments that apicoplast DNA Gyrase A and B, and Prex are required for apicoplast genome replication and growth of the parasite. Analysis of apicoplast genome replication by structured illumination microscopy in T. gondii tachyzoites showed that apicoplast nucleoid division and segregation initiate at the beginning of S phase and conclude during mitosis. Thus, the replication and division of the apicoplast nucleoid is highly coordinated with nuclear genome replication and mitosis. Our observations highlight essential components of apicoplast genome maintenance and shed light on the timing of this process in the context of the overall parasite cell cycle.

Recent Progress in Small Molecular Inhibitors of DNA Gyrase.

In the past few decades, with the abuse of antibiotics, bacterial resistance has enhanced constantly. More and more super species of bacteria, which are seriously threatening human health, have been discovered. Developing novel antibacterial agents to overcome the drug-resistance is an urgent duty. We all know that blocking the information-transfer of bacterial DNA and RNA is one of the effective ways to inhibit bacterial growth. Therefore, as the indispensable enzyme for DNA replication and transcription, DNA gyrase is one of the important targets for bacterial inhibitors. Accordingly, many inhibitors of DNA gyrase have also been developed. In this review, to highlight the recent progress in DNA gyrase inhibitors, the study in this field over the past three years (2017-2019) was summarized and organized based on their backbones or core moieties. Both of the subunits of DNA gyrase were taken into consideration. These DNA gyrase inhibitors have been classified based on their backbones or core moieties. After the comparison of the divided 14 categories, we could achieve some clues for future modification. In particular, we found that benzodiazepines and naphthalene heterocycles were the most common structures in the drug design. On the other hand, isopropyl and cyclopropyl have also been used in drug design, which provides more inspiration for the investigations. Except for GSK2140944, which has entered the phase III clinical trial stage, other compounds here were not fully promulgated with their optimal pharmacokinetic activity. We briefly summed up the current situation and future challenges on this topic. Through the discussion of the design strategies and drug effect, we hope that this review can provide a focused direction for future researches.

Benzoic and salicylic acid are the signaling molecules of Chlorella cells for improving cell growth

Cell-to-cell communication regulates microalgae production via signaling molecules (SMs), but few microalgal SM species are known. Here, we document two new microalgae SMs, benzoic acid (BA) and salicylic acid (SA). Initially, crude SMs were extracted from a microalgae culture in which microalgae grew on heterotrophic-enriched phosphorus nutrition. The extracted SMs enhanced Chlorella growth by ∼72%, promoted nutrient uptake, and up-regulated the mitogen-activated protein-kinase signaling cascade. Fourier transform infrared and nuclear magnetic resonance analyses identified the putative SMs was aromatic carboxylic acids. BA and SA were identified using high-resolution mass spectrometry. BA and SA addition increased cell growth by ∼75% and ∼25%; and improved ATP production by ∼35% and ∼20%. Transcriptomic analysis showed that BA and SA were biosynthesized via CoA-dependent, non-oxidative pathway. The SMs upregulated TCA-cycle enzymes, which promoted carbon assimilation and activated DNA-replicating enzyme, so that accelerated cell division. This study identified two new SMs for microalgae cell communication and provides means to identify other SMs.