Asgard ESCRT-III and VPS4 reveal evolutionary conserved chromatin binding properties of the ESCRT machinery

The ESCRT machinery drive membrane remodeling in numerous processes in eukaryotes. Genes encoding for ESCRT proteins have been identified in Asgard archaea, a newly discovered superphylum, currently recognized as the ancestor of all eukaryotes. This begs the question of the functional evolutionary origin of this machinery and its conservation across lineages. Here, we find that Asgard-ESCRTs exhibit conserved DNA-binding properties, which is derived from recruitment of specific members. We show that Asgard-ESCRT-III/VPS4 homologs interact with one another inside mammalian cells, associate with chromatin, and recruit their counterparts to organize in discrete foci in the mammalian nucleus. This is congruent with human-ESCRT-III homologs. We find that human- and Asgard-ESCRT-IIIs associate with chromatin via the same N terminal domain, and that human-ESCRT-III can recruit Asgard-VPS4 to the nucleus to form foci. Therefore, ESCRTs possess chromatin binding properties that were preserved through the billion years of evolution that separate Asgard and human cells.

Alkaloid Escholidine and Its Interaction with DNA Structures

Berberine, the most known quaternary protoberberine alkaloid (QPA), has been reported to inhibit the SIK3 protein connected with breast cancer. Berberine also appears to reduce the bcl-2 and XIAP expression-proteins responsible for the inhibition of apoptosis. As some problems in the therapy with berberine arose, we studied the DNA binding properties of escholidine, another QPA alkaloid. CD, fluorescence, and NMR examined models of i-motif and G-quadruplex sequences present in the n-myc gene and the c-kit gene. We provide evidence that escholidine does not induce stabilization of the i-motif sequences, while the interaction with G-quadruplex structures appears to be more significant.

Ubiquitin-specific proteases UBP12 and UBP13 promote shade avoidance response by enhancing PIF7 stability

Changes in light quality caused by the presence of neighbor proximity regulate many growth and development processes of plants. PHYTOCHROME INTERACTING FACTOR 7 (PIF7), whose subcellular localization, DNA-binding properties, and protein abundance are regulated in a photoreversible manner, plays a central role in linking shade light perception and growth responses. How PIF7 activity is regulated during shade avoidance responses has been well studied, and many factors involved in this process have been identified. However, the detailed molecular mechanism by which shade light regulates the PIF7 protein level is still largely unknown. Here, we show that the PIF7 protein level regulation is important for shade-induced growth. Two ubiquitin-specific proteases, UBP12 and UBP13, were identified as positive regulators in shade avoidance responses by increasing the PIF7 protein level. The ubp12-2w/13–3 double mutant displayed significantly impaired sensitivity to shade-induced cell elongation and reproduction acceleration. Our genetic and biochemical analysis showed that UBP12 and UBP13 act downstream of phyB and directly interact with PIF7 to maintain PIF7 stability and abundance through deubiquitination.

1052. Characterisation of the DNA binding properties of ridinilazole, a selective antibiotic currently in phase III trials for the treatment of Clostridioides difficile

Background Clostridioides difficile infection (CDI) is recognised by the CDC as an “urgent threat” in the USA, responsible for nearly 13,000 deaths, and carries an economic burden ranging from &5.4 to &6.3 billion per year. In a phase II study, ridinilazole was shown to be effective at treating CDI and decreasing subsequent recurrence compared to vancomycin. However, the precise mechanism of action of ridinilazole has yet to be fully elucidated. We now present data that reveals ridinilazole clearly co-localises with DNA in C. difficile and binds with high affinity to the minor groove of DNA. These interactions are predicted to have consequences on cellular functions within C. difficile. Methods High resolution confocal microscopy was used to track the intracellular localisation of ridinilazole in C. difficile. Fluorescence intensity was used to characterise the DNA binding properties of ridinilazole; sequence specificity was demonstrated with AT- or GC-rich DNA polymers, and tight binding was shown using short double-stranded oligonucleotides. Hanging drop vapour diffusion enabled co-crystallisation and subsequent structural determination of DNA-bound ridinilazole. Results Confocal microscopy revealed clear co-localisation of ridinilazole to the DNA within C. difficile. Ridinilazole demonstrated a dose-dependent increase in fluorescence in response to increasing concentration of target DNA. Fluorescence binding studies revealed that ridinilazole shows a preference towards AT-rich DNA sequences. Tight binding characteristics were demonstrated by ridinilazole in complex with short double-stranded oligonucleotides, returning dissociation constants (Kd) of 20 – 50 nM. Crystallisation enabled co-structures of ridinilazole bound to the minor groove of double-stranded DNA oligonucleotides to be solved. Conclusion Ridinilazole demonstrates tight binding with sequence specificity within the minor groove of DNA and co-localises with DNA in C. difficle. Further analysis is ongoing to fully understand this novel mechanism of action, the downstream consequences of these interactions and how they contribute to the bactericidal activity of ridinilazole. Disclosures Clive Mason, PhD, Summit Therapeutics (Employee, Shareholder) Tim Avis, n/a, Summit therapeutics (Shareholder) Chris Coward, PhD, Summit Therapeutics (Employee, Scientific Research Study Investigator, Shareholder) David Powell, PhD, Summit Therapeutics (Employee) Kevin W. Garey, Pharm.D., M.S., FASHP, Summit Therapeutics (Research Grant or Support)

Antibacterial Activity and DNA Binding Properties of Bivalent Metal Complexes of Cuminaldehyde Acetoylhydrazone

Metallo-hydrazones having the formula [M(IBAH)2] (where, M = Ni(II), Cu(II) and Zn(II); IBAH = p-Isopropylbenzaldehyde acetoylhydrazone) are prepared and confirmed on the basis of physico-chemical and spectral analyses. Conductivity data revealed that the complexes are non-electrolytes. Metal-DNA interactions are investigated using absorption spectrophotometry. Binding constant (Kb) data revealed that the copper complex interact DNA more strongly than other complexes. Antibacterial activity studies indicated higher activity for complexes than the metal free hydrazone ligand. The copper compound displays higher activity. DNA binding constants are correlated with the activity of metal compounds in this article.