Biology subjects

Verga, D.

Publications and source records attributed to Verga, D..

4 recordsLinked to original sources

bioRxiv2024

G-quadruplexes in Haloferax volcanii

Archaea, a domain of microorganisms found in diverse environments including the human microbiome, represent the closest known prokaryotic relatives of eukaryotes. This phylogenetic proximity positions them as a relevant model for investigating the evolutionary origins of nucleic acid secondary structures such as G-quadruplexes (G4s), which play regulatory roles in transcription and replication. Although G4s have been extensively studied in eukaryotes, their presence and function in archaea remain poorly characterized. In this study, a genome-wide analysis of the halophilic archaeon Haloferax volcanii identified over 5, 800 potential G4-forming sequences. Biophysical validation confirmed that many of these sequences adopt stable G4 conformations in vitro. Using G4-specific detection tools and super-resolution microscopy, G4 structures were visualized in vivo in both DNA and RNA across multiple growth phases. Comparable findings were observed in the thermophilic archaeon Thermococcus barophilus. Functional analysis using helicase-deficient H. volcanii strains further identified candidate enzymes involved in G4 resolution. These results establish H. volcanii as a tractable archaeal model for G4 biology.

molecular biology↗

bioRxiv2023

G-quadruplexes are promoter elements controlling nucleosome exclusion and RNA polymerase II pausing

Despite their central role in transcription, it has been difficult to define universal sequences associated to eukaryotic promoters. Within chromatin context, recruitment of the transcriptional machinery requires opening of the promoter but how DNA elements could contribute to this process has remained elusive. Here, we show that G-quadruplex (G4) secondary structures are highly enriched mammalian core promoter elements. G4s are located at the deepest point of nucleosome exclusion at promoters and correlate with maximum promoter activity. We found that experimental G4s exclude nucleosomes both in vivo and in vitro and display a strong positioning potential. At model promoters, impairing G4s affected both transcriptional activity and chromatin opening. G4 destabilization also resulted in an inactive promoter state and affected transition to effective RNA production in live imaging experiments. Finally, G4 stabilization resulted in global reduction of proximal promoter pausing. Altogether, our data introduce G4s as bona fide promoter elements allowing nucleosome exclusion and facilitating pause release by the RNA Polymerase II.

genomics↗

bioRxiv2019

G-quadruplexes sequester free heme in living cells

Heme is an essential cofactor for many enzymes, but free heme is toxic and its levels are tightly regulated. G-quadruplexes bind heme avidly in vitro, raising the possibility that they may sequester heme in vivo. If so, then treatment that displaces heme from quadruplexes is predicted to induce expression of genes involved in iron and heme homeostasis. Here we show that PhenDC3, a G-quadruplex ligand structurally unrelated to heme, displaces quadruplex-bound heme in vitro and alters transcription in cultured human cells, up-regulating genes that support heme degradation and iron homeostasis, and most strikingly causing a 30-fold induction of heme oxidase 1, the key enzyme in heme degradation. We propose that G-quadruplexes sequester heme to protect cells from the pathophysiological consequences of free heme. This identifies a new function for G-quadruplexes and a new mechanism for protection of cells from heme.

genomics↗

bioRxiv2019

Probing Ligand and Cation Binding Sites in G-Quadruplex Nucleic Acids by Mass Spectrometry and Electron Photodetachment Dissociation Sequencing

Mass spectrometry provides exquisite detail on ligand and cation binding stoichiometries with a DNA target. The next important step is to develop reliable methods to determine the cation and ligand binding sites in each complex separated by the mass spectrometer. To circumvent the caveat of ligand derivatization for cross-linking, which may alter the ligand binding mode, we explored a tandem mass spectrometry (MS/MS) method that does not require ligand derivatization, and is therefore also applicable to localize metal cations. By obtaining more negative charge states for the complexes using supercharging agents, and by creating radical ions by electron photodetachment, oligonucleotide bonds become weaker than the DNA-cation or DNA-ligand noncovalent bonds upon collision-induced dissociation of the radicals. This electron photodetachment (EPD) method allows to locate the binding regions of cations and ligands by top-down sequencing of the oligonucleotide target. The very potent G-quadruplex ligands 360A and PhenDC3 were found to replace a potassium cation and bind close to the central loop of 4-repeat human telomeric sequences.

biophysics↗