Thus, while it is clear that Top1 regulates R-loops and prevents R-loop-induced genomic instability, the range of loci that are sensitive to R-loop modulation by Top1 is not known. Finally, stabilization of Top1cc by Top1 inhibitors such as camptothecin and its derivatives leads to R-loop stabilization in human cells upon short treatment and to transcription-dependent DNA breakage that can be partially suppressed by RNase H expression. Furthermore, persistent depletion of Top1 in mammalian cells leads to replicative stress and replication-transcription conflicts that can be rescued by overexpression of RNase H.
Indeed, deletion of the bacterial topA gene, an enzyme that only relaxes negative supercoils, creates R-loop-prone hypernegatively supercoiled DNA and causes a growth defect that can be suppressed by over-expression of Ribonuclease H (RNase H), an enzyme that degrades RNA strands in RNA:DNA hybrids. The relaxation activity on negative supercoils is thought to reduce co-transcriptional R-loop formation which in turns prevents replication / transcription interference and favors genome stability. Top1 activity can relax negative supercoils by cutting one of the DNA strands, creating a transient Top1-DNA cleavage complex (Top1cc), and performing a controlled rotation of the cut strand around the uncut strand. DNA Topoisomerase I (Top1) is one main cellular factor controlling topological homeostasis. Negative supercoiling generated behind the elongating RNA polymerase is thought to facilitate R-loop formation by inducing an underwound DNA state favorable to the re-annealing of the nascent transcript. Mapping data indicate that these non-B DNA structures are prevalent in mammalian genomes, where they form dynamically over conserved regions. R-loops are formed during transcription upon reannealing of the nascent transcript to the DNA template strand, forming an RNA:DNA hybrid and forcing the non-template strand to loop out. R-loop structures, a prevalent non-B DNA structure in mammalian genomes, have been particularly linked to genomic instability by causing interference between the replication and transcription machineries. Our 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.īiological processes such as transcription and replication generate torsional stress on the DNA double helix that, if not properly dealt with, can lead to genome instability. Interestingly, Top1 depletion coincides with a block of the cell cycle in G0/G1 phase and a trend towards replication delay. R-loop losses, by contrast, occur in gene-rich regions overlapping H3K27me3-marked active replication initiation regions.
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. Here, 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.
How Top1 regulates R-loop structures at a global level is unknown. DNA Topoisomerase I (Top1) is often thought to regulate R-loop formation owing to its ability to resolve both positive and negative supercoils. Co-transcriptional R-loops are abundant non-B DNA structures in mammalian genomes.