NHEJ consists in the direct ligation of the two DNA ends, and is mediated by the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV-XRCC4 complexes.
HR relies on a homologous template for repair, is mediated by a large number of proteins, including RAD51, and is stimulated by the activation of two kinases, namely ATM (ataxia telangiectasia mutated) kinase at DSBs, and ATR (ATM and Rad3-related) kinase at stalled replication forks.
DSBs are the most dangerous and deleterious of all lesions, and two major pathways exist to repair them: homologous recombination (HR) and non-homologous end joining (NHEJ) ( 4–5). A complex mechanism, known as the DNA damage response (DDR), exists in higher organisms to sense damage, block the cell cycle and repair DNA lesions via a large variety of mechanisms. Lesions, such as modifications and loss of bases or replication errors, interfer with DNA transactions (i.e. replication and transcription) and induce DNA damage signalling and single- (SSB) and double-strand breaks (DSB) ( 1–3). This wealth of data was exploited to design an efficient synthetic lethality strategy combining dual ligands with clinically relevant DDR inhibitors.ĭNA is constantly damaged by exogeneous and endogenous processes. We designed and used a clickable ligand, TrisNP-α, to provide unique insights into the TWJ landscape in cells and its modulation upon co-treatments. We show here that cytotoxic activity of TWJ-/G4-ligands is mitigated by the DNA damage response (DDR) and DNA topoisomerase 2 (TOP2), making them different from typical G4-ligands, and implying a pivotal role of TWJs in cells. The DNA binding modes of these new, dual TWJ-/G4-ligands were studied by a panel of in vitro methods and theoretical simulations, and their cellular properties by extensive cell-based assays. While previous approaches have claimed to use ligands that specifically target either G4s or TWJs, we report here on a new approach in which ligands targeting both TWJs and G4s in vitro demonstrate cellular effects distinct from that of G4 ligands, and attributable to TWJ targeting. G4s and TWJs can be stabilised by small molecules (ligands) that have high chemotherapeutic potential, either as standalone DNA damaging agents or combined in synthetic lethality strategies.
DNA is intrinsically dynamic and folds transiently into alternative higher-order structures such as G-quadruplexes (G4s) and three-way DNA junctions (TWJs).