cx-5461 and pyridostatin

Guanine-rich DNA can fold into secondary structures known as G-quadruplexes (G4s). G4s can form from a single DNA strand (intramolecular) or from multiple DNA strands (intermolecular), but studies on their biological functions have been often limited to intramolecular G4s, owing to the low probability of intermolecular G4s to form within genomic DNA. Herein, we report the first example of an endogenous protein, Cockayne Syndrome B (CSB), that can bind selectively with picomolar affinity toward intermolecular G4s formed within rDNA while displaying negligible binding toward intramolecular structures. We observed that CSB can selectively resolve intermolecular over intramolecular G4s, demonstrating that its selectivity toward intermolecular structures is also reflected at the resolvase level. Immunostaining of G4s with the antibody BG4 in CSB-impaired cells (CS1AN) revealed that G4-staining in the nucleolus of these cells can be abrogated by transfection of viable CSB, suggesting that intermolecular G4s can be formed within rDNA and act as binding substrate for CSB. Given that loss of function of CSB elicits premature aging phenotypes, our findings indicate that the interaction between CSB and intermolecular G4s in rDNA could be of relevance to maintain cellular homeostasis.

Topics: Aminoquinolines; Animals; Benzothiazoles; Cell Nucleolus; DNA; DNA Helicases; DNA Repair Enzymes; G-Quadruplexes; HeLa Cells; Humans; Naphthyridines; Picolinic Acids; Poly-ADP-Ribose Binding Proteins; Protein Binding; Recombinases; Sf9 Cells; Spodoptera

G-quadruplexes (G4) are non-canonical DNA structures found in the genome of most species including human. Small molecules stabilizing these structures, called G4 ligands, have been identified and, for some of them, shown to induce cytotoxic DNA double-strand breaks. Through the use of an unbiased genetic approach, we identify here topoisomerase 2α (TOP2A) as a major effector of cytotoxicity induced by two clastogenic G4 ligands, pyridostatin and CX-5461, the latter molecule currently undergoing phase I/II clinical trials in oncology. We show that both TOP2 activity and transcription account for DNA break production following G4 ligand treatments. In contrast, clastogenic activity of these G4 ligands is countered by topoisomerase 1 (TOP1), which limits co-transcriptional G4 formation, and by factors promoting transcriptional elongation. Altogether our results support that clastogenic G4 ligands act as DNA structure-driven TOP2 poisons at transcribed regions bearing G4 structures.

Topics: Aminoquinolines; Antineoplastic Agents; Benzothiazoles; Cell Line; Cell Proliferation; Cell Survival; Colony-Forming Units Assay; DNA Breaks, Double-Stranded; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; G-Quadruplexes; Gene Expression Regulation, Enzymologic; Humans; Naphthyridines; Picolinic Acids; Poly-ADP-Ribose Binding Proteins; Polymorphism, Single Nucleotide; RNA Interference; RNA-Seq