Summary R-loops, consisting of an RNA-DNA hybrid and displaced single-stranded DNA, are physiological structures that regulate various cellular processes occurring on chromatin. Intriguingly, changes in R-loop dynamics have also been associated with DNA damage accumulation and genome instability, however the mechanisms underlying R-loop induced DNA damage remain unknown. Here we demonstrate in human cells that R-loops induced by the absence of diverse RNA processing factors, including the RNA/DNA helicases Aquarius (AQR) and Senataxin (SETX), or by the inhibition of topoisomerase I, are actively processed into DNA double-strand breaks (DSBs) by the nucleotide excision repair endonucleases XPF and XPG. Surprisingly, DSB formation requires the transcription-coupled nucleotide excision repair (TC-NER) factor Cockayne syndrome group B (CSB), but not the global genome repair protein XPC. These findings reveal an unexpected and potentially deleterious role for TC-NER factors in driving R-loop-induced DNA damage and genome instability.

The Ubc9 SUMO-conjugating enzyme and the Siz1 SUMO ligase sumoylate several repair and recombination proteins, including PCNA. Sumoylated PCNA binds Srs2, a helicase counteracting certain recombination events. Here we show that ubc9 mutants depend on checkpoint, recombination, and replication genes for growth. ubc9 cells maintain stalled-fork stability but exhibit a Rad51-dependent accumulation of cruciform structures during replication of damaged templates. Mutations in the Mms21 SUMO ligase resemble the ubc9 mutations. However, siz1, srs2, or pcna mutants altered in sumoylation do not exhibit the ubc9/mms21 phenotype. Like ubc9/mms21 mutants, sgs1 and top3 mutants also accumulate X molecules at damaged forks, and Sgs1/BLM is sumoylated. We propose that Ubc9 and Mms21 act in concert with Sgs1 to resolve the X structures formed during replication. Our results indicate that Ubc9- and Mms21-mediated sumoylation functions as a regulatory mechanism, different from that of replication checkpoints, to prevent pathological accumulation of cruciform structures at damaged forks.

R-loops, nucleic acid structures consisting of an RNA-DNA hybrid and displaced single-stranded DNA, are ubiquitous in organisms from bacteria to mammals. First described in bacteria where they initiate DNA replication, it now appears that R-loops regulate diverse cellular processes such as gene expression, immunoglobulin class switching and DNA repair. Changes in R-loop regulation induce DNA damage and genome instability, and recently it was shown that R-loops are associated with neurodegenerative disorders. Here, we discuss recent developments in the field; in particular, the regulation and effects of R-loops in cells, their effect on genomic and epigenomic stability and their potential contribution to the origin of certain diseases, including cancer and neurodegenerative disorders.

The hormone estrogen (E2) binds the estrogen receptor to promote transcription of E2-responsive genes in the breast and other tissues. E2 also has links to genomic instability, and elevated E2 levels are tied to breast cancer. Here, we show that E2 stimulation causes a rapid, global increase in the formation of R-loops, co-transcriptional RNA-DNA products, which in some instances have been linked to DNA damage. We show that E2-dependent R-loop formation and breast cancer rearrangements are highly enriched at E2-responsive genomic loci and that E2 induces DNA replication-dependent double-strand breaks (DSBs). Strikingly, many DSBs that accumulate in response to E2 are R-loop dependent. Thus, R-loops resulting from the E2 transcriptional response are a significant source of DNA damage. This work reveals a novel mechanism by which E2 stimulation leads to genomic instability and highlights how transcriptional programs play an important role in shaping the genomic landscape of DNA damage susceptibility.DOI: http://dx.doi.org/10.7554/eLife.17548.001