G quadruplexes (G4s) and R loops are noncanonical DNA structures that can regulate basic nuclear processes and trigger DNA damage, genome instability, and cell killing. By different technical approaches, we here establish that specific G4 ligands stabilize G4s and simultaneously increase R-loop levels within minutes in human cancer cells. Genome-wide mapping of R loops showed that the studied G4 ligands likely cause the spreading of R loops to adjacent regions containing G4 structures, preferentially at 3′-end regions of expressed genes, which are partially ligand-specific. Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4 ligands in BRCA2-proficient and BRCA2-silenced cancer cells. Moreover, even if the studied G4 ligands increased noncanonical DNA structures at similar levels in nuclear chromatin, their cellular effects were different in relation to cell-killing activity and stimulation of micronuclei, a hallmark of genome instability. Our findings therefore establish that G4 ligands can induce DNA damage by an R loop-dependent mechanism that can eventually lead to different cellular consequences depending on the chemical nature of the ligands.

Summary DNA double-strand break (DSB) repair is mediated by multiple pathways. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a multiplexed reporter assay in combination with Cas9 cutting, we systematically measure the relative activities of three DSB repair pathways as a function of chromatin context in >1,000 genomic locations. This reveals that non-homologous end-joining (NHEJ) is broadly biased toward euchromatin, while the contribution of microhomology-mediated end-joining (MMEJ) is higher in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 reverts the balance toward NHEJ. Single-stranded template repair (SSTR), often used for precise CRISPR editing, competes with MMEJ and is moderately linked to chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance and guidance for the design of Cas9-mediated genome editing experiments.

BackgroundCo-transcriptional R-loops are abundant non-B DNA structures in mammalian genomes. DNA Topoisomerase I (Top1) is often thought to regulate R-loop formation owing to its ability to resolve both positive and negative supercoils. How Top1 regulates R-loop structures at a global level is unknown.ResultsHere, we perform high-resolution strand-specific R-loop mapping in human cells depleted for Top1 and find that Top1 depletion results in both R-loop gains and losses at thousands of transcribed loci, delineating two distinct gene classes. R-loop gains are characteristic for long, highly transcribed, genes located in gene-poor regions anchored to Lamin B1 domains and in proximity to H3K9me3-marked heterochromatic patches. R-loop losses, by contrast, occur in gene-rich regions overlapping H3K27me3-marked active replication initiation regions. Interestingly, Top1 depletion coincides with a block of the cell cycle in G0/G1 phase and a trend towards replication delay.ConclusionsOur findings reveal new properties of Top1 in regulating R-loop homeostasis in a context-dependent manner and suggest a potential role for Top1 in modulating the replication process via R-loop formation.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1478-1) contains supplementary material, which is available to authorized users.