RAD51 Inhibition Induces R-Loop Formation in Early G1 Phase of the Cell Cycle

R-loops are three-stranded structures generated by annealing of nascent transcripts to the template DNA strand, leaving the non-template DNA strand exposed as a single-stranded loop. Although R-loops play important roles in physiological processes such as regulation of gene expression, mitochondrial DNA replication, or immunoglobulin class switch recombination, dysregulation of the R-loop metabolism poses a threat to the stability of the genome. A previous study in yeast has shown that the homologous recombination machinery contributes to the formation of R-loops and associated chromosome instability. On the contrary, here, we demonstrate that depletion of the key homologous recombination factor, RAD51, as well as RAD51 inhibition by the B02 inhibitor did not prevent R-loop formation induced by the inhibition of spliceosome assembly in human cells. However, we noticed that treatment of cells with B02 resulted in RAD51-dependent accumulation of R-loops in an early G1 phase of the cell cycle accompanied by a decrease in the levels of chromatin-bound ORC2 protein, a component of the pre-replication complex, and an increase in DNA synthesis. Our results suggest that B02-induced R-loops might cause a premature origin firing.

Automated summary

R-loops are three-stranded structures formed by annealing of nascent transcripts and template DNA, playing important roles in physiological processes. Dysregulation of R-loop metabolism may pose a threat to genome stability. Depletion of RAD51 and inhibition by B02 did not prevent R-loop formation in human cells, while B02 treatment may lead to premature origin firing caused by RAD51-dependent accumulation of R-loops.