Chromatin Architectural Factors as Safeguards against Excessive Supercoiling during DNA Replication

Key DNA transactions, such as genome replication and transcription, rely on the speedy translocation of specialized protein complexes along a double-stranded, right-handed helical template. Physical tethering of these molecular machines during translocation, in conjunction with their internal architectural features, generates DNA topological strain in the form of template supercoiling. It is known that the build-up of transient excessive supercoiling poses severe threats to genome function and stability and that highly specialized enzymes—the topoisomerases (TOP)—have evolved to mitigate these threats. Furthermore, due to their intracellular abundance and fast supercoil relaxation rates, it is generally assumed that these enzymes are sufficient in coping with genome-wide bursts of excessive supercoiling. However, the recent discoveries of chromatin architectural factors that play important accessory functions have cast reasonable doubts on this concept. Here, we reviewed the background of these new findings and described emerging models of how these accessory factors contribute to supercoil homeostasis. We focused on DNA replication and the generation of positive (+) supercoiling in front of replisomes, where two accessory factors—GapR and HMGA2—from pro- and eukaryotic cells, respectively, appear to play important roles as sinks for excessive (+) supercoiling by employing a combination of supercoil constrainment and activation of topoisomerases. Looking forward, we expect that additional factors will be identified in the future as part of an expanding cellular repertoire to cope with bursts of topological strain. Furthermore, identifying antagonists that target these accessory factors and work synergistically with clinically relevant topoisomerase inhibitors could become an interesting novel strategy, leading to improved treatment outcomes.

TOP2A substitution enhances topoisomerase activity and causes transcriptional dysfunction in glioblastoma patients

High grade gliomas (HGGs) are aggressive, primary brain tumors with poor clinical outcomes. To better understand glioma pathobiology and find potential therapeutic susceptibilities, we designed a custom-panel 664 cancer- and epigenetics-related genes and employed targeted next generation sequencing to study the genomic landscape of somatic and germline variants in 182 glioma samples of different malignancy grades. Besides known alterations in TP53, IDH1, ATRX, EGFR genes, we found several novel variants that can be potential drivers in gliomas. In four patients from the Polish glioma cohort, we identified a novel recurrent mutation in the TOP2A gene coding for Topoisomerase 2A in glioblastomas (GBM, WHO grade IV gliomas). The mutation results in a substitution of glutamic acid (E) 948 to glutamine (Q) of TOP2 A and we predicted this E948Q substitution may affect DNA binding and a TOP2A enzymatic activity. Topoisomerases are enzymes that control the higher order DNA structure by introducing transient breaks and rejoining DNA strands. Using recombinant proteins we demonstrated stronger DNA binding and DNA supercoil relaxation activities of the variant proteins. Glioblastoma (GBM) patients with the mutated TOP2A had shorter overall survival than wild type TOP2A GBM patients. Computational analyses of transcriptomic data showed that the GBM samples with the mutated TOP2A have different transcriptomic patterns suggesting higher transcriptomic activity. The results suggest that TOP2A E948Q variant strongly binds to DNA and is more active in comparison to the wild-type protein. Altogether, our findings suggest that the E948Q substitution leads to gain of function by TOP2A.

Modulated control of DNA supercoiling balance by the DNA-wrapping domain of bacterial gyrase

Negative supercoiling by DNA gyrase is essential for maintaining chromosomal compaction, transcriptional programming, and genetic integrity in bacteria. Questions remain as to how gyrases from different species have evolved profound differences in their kinetics, efficiency, and extent of negative supercoiling. To explore this issue, we analyzed homology-directed mutations in the C-terminal, DNA-wrapping domain of the GyrA subunit of Escherichia coli gyrase (the ‘CTD’). The addition or removal of select, conserved basic residues markedly impacts both nucleotide-dependent DNA wrapping and supercoiling by the enzyme. Weakening CTD–DNA interactions slows supercoiling, impairs DNA-dependent ATP hydrolysis, and limits the extent of DNA supercoiling, while simultaneously enhancing decatenation and supercoil relaxation. Conversely, strengthening DNA wrapping does not result in a more extensively supercoiled DNA product, but partially uncouples ATP turnover from strand passage, manifesting in futile cycling. Our findings indicate that the catalytic cycle of E. coli gyrase operates at high thermodynamic efficiency, and that the stability of DNA wrapping by the CTD provides one limit to DNA supercoil introduction, beyond which strand passage competes with ATP-dependent supercoil relaxation. These results highlight a means by which gyrase can evolve distinct homeostatic supercoiling setpoints in a species-specific manner.

Ozenoxacin: A Novel Drug Discovery for the Treatment of Impetigo

Objective: Ozenoxacin is one of the potent quinolone antibiotics, recently approved by the United States Food and Drug Administration (USFDA) with reported pharmacology to treat the impetigo. The demand for better acting topical formulation is increasing day by day. The present review is an attempt to summarize the facts behind the chemistry and biological applications of Ozenoxacin. Mechanism of Action: This novel drug being a quinolone antibiotic compound, acts by inhibiting DNA gyrase A and topoisomerase IV and affects supercoiling, supercoil relaxation, chromosomal condensation, chromosomal decatenation and many others. Mechanism of Action: This novel drug being a quinolone antibiotic compound, acts by inhibiting DNA gyrase A and topoisomerase IV and affects supercoiling, supercoil relaxation, chromosomal condensation, chromosomal decatenation and many others. Pharmacology: Ozenoxacin has demonstrated to have a bactericidal activity against organisms, such as Staphylococcus aureus and Staphylococcus pyogenes. Ozenoxacin is non-fluorinated quinolone and being developed for the other dermatological bacterial infections as well. No sign of genotoxicity was observed when tested experimentally. Conclusion: The present review also covers the complete picture of pharmacokinetics, clinical trials, toxicity and future scope and possible avenues in this arena.

Single-molecule imaging of DNA gyrase activity in livingEscherichia coli

ABSTRACTBacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in liveEscherichia colicells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with ∼12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of ∼2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was ∼8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.