Overexpression of the Replicative Helicase in Escherichia coli Inhibits Replication Initiation and Replication Fork Reloading

Replicative helicases play central roles in chromosome duplication and their assembly onto DNA is regulated via initiators and helicase loader proteins. The Escherichia coli replicative helicase DnaB and the helicase loader DnaC form a DnaB6-DnaC6 complex that is required for loading DnaB onto single-stranded DNA. Overexpression of dnaC inhibits replication by promoting continual rebinding of DnaC to DnaB and consequent prevention of helicase translocation. Here we show that overexpression of dnaB also inhibits growth and chromosome duplication. This inhibition is countered by co-overexpression of wild-type DnaC but not of a DnaC mutant that cannot interact with DnaB, indicating that a reduction in DnaB6-DnaC6 concentration is responsible for the phenotypes associated with elevated DnaB concentration. Partial defects in the oriC-specific initiator DnaA and in PriA-specific initiation away from oriC during replication repair sensitise cells to dnaB overexpression. Absence of the accessory replicative helicase Rep, resulting in increased replication blockage and thus increased reinitiation away from oriC, also exacerbates DnaB-induced defects. These findings indicate that elevated levels of helicase perturb replication initiation not only at origins of replication but also during fork repair at other sites on the chromosome. Thus, imbalances in levels of the replicative helicase and helicase loader can inhibit replication both via inhibition of DnaB6-DnaC6 complex formation with excess DnaB, as shown here, and promotion of formation of DnaB6-DnaC6 complexes with excess DnaC [Allen GC, Jr., Kornberg A. Fine balance in the regulation of DnaB helicase by DnaC protein in replication in Escherichia coli. J. Biol. Chem. 1991;266:22096-22101; Skarstad K, Wold S. The speed of the Escherichia coli fork in vivo depends on the DnaB:DnaC ratio. Mol. Microbiol. 1995;17:825-831]. Thus, there are two mechanisms by which an imbalance in the replicative helicase and its associated loader protein can inhibit genome duplication

Mu Insertions Are Repaired by the Double-Strand Break Repair Pathway of Escherichia coli

Mu is both a transposable element and a temperate bacteriophage. During lytic growth, it amplifies its genome by replicative transposition. During infection, it integrates into the Escherichia coli chromosome through a mechanism not requiring extensive DNA replication. In the latter pathway, the transposition intermediate is repaired by transposase-mediated resecting of the 5′ flaps attached to the ends of the incoming Mu genome, followed by filling the remaining 5 bp gaps at each end of the Mu insertion. It is widely assumed that the gaps are repaired by a gap-filling host polymerase. Using the E. coli Keio Collection to screen for mutants defective in recovery of stable Mu insertions, we show in this study that the gaps are repaired by the machinery responsible for the repair of double-strand breaks in E. coli—the replication restart proteins PriA-DnaT and homologous recombination proteins RecABC. We discuss alternate models for recombinational repair of the Mu gaps

Genetic studies of replication restart in Escherichia coli

Faithful DNA replication is essential for all organisms to maintain genetic integrity. During the DNA replication, replications forks are frequently stalled or collapsed due to the encounter of DNA lesions or blocking proteins. These events can occur anywhere on the chromosome which is away from the origin of replication. For survival, cells require a number of proteins to repair the damages and restart the replication near or at the damaged site. In Escherichia coli, a group of proteins called primosomal proteins consisting of PriA, PriB, PriC, Rep, DnaT, DnaC, DnaB and DnaG are required for directing DnaB replicative helicase back onto DNA substrates. The main difference between chromosomal replication at oriC and replication restart is the former process is initiated by DnaA recognizing a specific sequence of oriC while the latter process can be initiated by PriA recognizing a specific DNA structure. The mechanism of replication restart is highly ordered and well regulated, and to date, this mechanism has yet to be fully understood. This lab uses a genetic tool to understand replication restart in vivo and discovered that replication restart in E. coli can be explained by a multiple replication restart pathway model (Sandler, 2000). This dissertation presents work that advances our knowledge of replication restart by studying Rep and PriB using a genetic approach. Although it has been shown that Rep and PriB are important for replication restart, the contribution of these two proteins in vivo is still not clearly understood. In the case of Rep, this study provided the characterization of three rep mutants: a rep null mutant, a rep defective in a regulation of Rep monomer helicase activity and a rep ATP hydrolysis deficient mutant. Although these rep mutants showed a similar phenotype, there were some differences such as the phenotypes when combined with other mutations and spontaneous suppressors. In this study, a rep null mutant exhibited an unexpected phenotype including high basal levels of SOS expression and cell filamentation when combined with priB. The priB rep double mutant developed a spontaneous suppressor mapped in dnaC region. A rep mutant defective in an autoinhibition of Rep monomer helicase activity showed a more detrimental phenotype than a rep null mutant when combined with priB suggesting that this mutant Rep inappropriately removes PriC and completes with PriA. The other rep mutant, an ATPase deficient mutant, showed a similar phenotype to that of a rep null mutant as a single mutant and a double mutant with priB, however, spontaneous suppressors developed in the regions close to priC and dnaC, but not in the either of these genes. Lastly, PriB mutants that biochemical evidence has shown some effects on PriA, ssDNA and DnaT binding were tested for effects on replication restart in vivo. The studies of these PriB mutants lead to a model of the regulation of replication restart via PriA-PriB pathway which can be explained by a hand-off mechanism for primosomal assembly

BIOACTIVE ACTIVITY OF A RECOMBINANT LONGAN (Dimocarpus longan LOUR.) SEED PEPTIDE

Background: Consumption of antioxidants has been evident to prevent diseases caused by free radicals damage. Antioxidants can be found in the form of peptide in various natural sources. From our previous study, to overcome obstacles of direct longan seed hydrolysate extraction, the recombinant Longan1 peptide which contains 4 repeats of ISYVVPVYIAEITPKTFRGGF linked by D was produced from Escherichia coli. The in vitro bioactive properties of this recombinant peptide were characterized.  Methods: The recombinant and chemically synthesized Longan1 peptides were tested for bioactive activity including, DPPH, ABTS, and nitric oxide radical scavenging assays, the ability to protect plasmid DNA from hydroxyl radicals, anti-proliferative activity to several cancer cell lines, and antiinflammatory effects in cell culture level. Results: The recombinant peptide revealed antioxidative activities, including DPPH, ABTS, and nitric oxide radical scavenging activity, which are similar to the chemically-synthesized one. However, the recombinant peptide exhibited higher in vitro ability to protect DNA from hydroxyl radicals. The IC50 value of the recombinant Longan1 peptide could only be calculated through the assay of antiproliferation of stomach KATO-III cancer cell line, while IC50 value from the chemically synthesized peptide could not be calculated in any tested cell lines. Finally, the anti-inflammatory effect determined by the inhibition of nitric oxide production from macrophages RAW 264.7 activated by LPS revealed that the recombinant Longan1 peptide could inhibit nitric oxide production from macrophage cells, whereas the chemically-synthesized one could not. Conclusion: With all these properties, the recombinant Longan1 peptide seems to have bioactivity that can possibly be a candidate for further medical application or supplementary products. &nbsp

A hand-off mechanism for primosome assembly in replication restart.

Collapsed DNA replication forks must be reactivated through origin-independent reloading of the replication machinery (replisome) to ensure complete duplication of cellular genomes. In E. coli, the PriA-dependent pathway is the major replication restart mechanism and requires primosome proteins PriA, PriB, and DnaT for replisome reloading. However, the molecular mechanisms that regulate origin-independent replisome loading are not fully understood. Here, we demonstrate that assembly of primosome protein complexes represents a key regulatory mechanism, as inherently weak PriA-PriB and PriB-DnaT interactions are strongly stimulated by single-stranded DNA. Furthermore, the binding site on PriB for single-stranded DNA partially overlaps the binding sites for PriA and DnaT, suggesting a dynamic primosome assembly process in which single-stranded DNA is handed off from one primosome protein to another as a repaired replication fork is reactivated. This model helps explain how origin-independent initiation of DNA replication is restricted to repaired replication forks, preventing over-replication of the genome

Incidence of Complications in Obese Patients Undergoing Elective Spine Surgery Under General Anesthesia: A Retrospective Study

Background The prevalence of obesity has been increasing worldwide. Simultaneously, the number of obese patients undergoing anesthesia and the risk of anesthesia-related complications are increasing. Our study aimed to identify the incidence of intraoperative anesthetic complications in obese patients undergoing elective spine surgery

The Design and Synthesis of a New Series of 1,2,3-Triazole-Cored Structures Tethering Aryl Urea and Their Highly Selective Cytotoxicity toward HepG2

Target cancer drug therapy is an alternative treatment for advanced hepatocellular carcinoma (HCC) patients. However, the treatment using approved targeted drugs has encountered a number of limitations, including the poor pharmacological properties of drugs, therapy efficiency, adverse effects, and drug resistance. As a consequence, the discovery and development of anti-HCC drug structures are therefore still in high demand. Herein, we designed and synthesized a new series of 1,2,3-triazole-cored structures incorporating aryl urea as anti-HepG2 agents. Forty-nine analogs were prepared via nucleophilic addition and copper-catalyzed azide-alkyne cycloaddition (CuAAC) with excellent yields. Significantly, almost all triazole-cored analogs exhibited less cytotoxicity toward normal cells, human embryonal lung fibroblast cell MRC-5, compared to Sorafenib and Doxorubicin. Among them, 2m’ and 2e exhibited the highest selectivity indexes (SI = 14.7 and 12.2), which were ca. 4.4- and 3.7-fold superior to that of Sorafenib (SI = 3.30) and ca. 3.8- and 3.2-fold superior to that of Doxorubicin (SI = 3.83), respectively. Additionally, excellent inhibitory activity against hepatocellular carcinoma HepG2, comparable to Sorafenib, was still maintained. A cell-cycle analysis and apoptosis induction study suggested that 2m’ and 2e likely share a similar mechanism of action to Sorafenib. Furthermore, compounds 2m’ and 2e exhibit appropriate drug-likeness, analyzed by SwissADME. With their excellent anti-HepG2 activity, improved selectivity indexes, and appropriate druggability, the triazole-cored analogs 2m’ and 2e are suggested to be promising candidates for development as targeted cancer agents and drugs used in combination therapy for the treatment of HCC

The amide derivative of anticopalic acid induces non-apoptotic cell death in triple-negative breast cancer cells by inhibiting FAK activation

Abstract Anticopalic acid (ACP), a labdane type diterpenoid obtained from Kaempferia elegans rhizomes, together with 21 semi-synthetic derivatives, were evaluated for their cancer cytotoxic activity. Most derivatives displayed higher cytotoxic activity than the parent compound ACP in a panel of nine cancer cell lines. Among the tested compounds, the amide 4p showed the highest cytotoxic activity toward leukemia cell lines, HL-60 and MOLT-3, with IC50 values of 6.81 ± 1.99 and 3.72 ± 0.26 µM, respectively. More interestingly, the amide derivative 4l exhibited cytotoxic activity with an IC50 of 13.73 ± 0.04 µM against the MDA-MB-231 triple-negative breast cancer cell line, which is the most aggressive type of breast cancer. Mechanistic studies revealed that 4l induced cell death in MDA-MB-231 cells through non-apoptotic regulated cell death. In addition, western blot analysis showed that compound 4l decreased the phosphorylation of FAK protein in a concentration-dependent manner. Molecular docking simulations elucidated that compound 4l could potentially inhibit FAK activation by binding to a pocket of FAK kinase domain. The data suggested that compound 4l could be a potential FAK inhibitor for treating triple-negative breast cancer and worth being further investigated