Transcription-Coupled Nucleotide Excision Repair Factors Promote R-Loop-Induced Genome Instability

Abstract

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.

Cdk-dependent phosphorylation regulates TRF1 recruitment to PML bodies and promotes C-circle production in ALT cells

“…We have shown that association of ( pT371)TRF1 with APBs is sensitive to RNaseH1 in the presence of camptothecin, an inhibitor of topoisomerase I, an enzyme known to prevent the formation of RNA-DNA hybrids and to resolve conflicts between transcription and replication (Hamperl and Cimprich, 2014;Tuduri et al, 2009). RNA-DNA hybrids can be processed into DNA double-strand breaks (DSBs) (Sollier et al, 2014), and recently, it has been reported that DNA DSBs at ALT telomeres induce telomere clustering, APB formation as well as other homologousrecombination-based ALT activities (Cho et al, 2014). Perhaps, telomeric RNA-DNA hybrids arising from transcription and replication collision might be a natural source of DNA damage that triggers ALT.…”

“…RNA-DNA hybrids, also known as R loops, can be processed to give rise to DNA doublestrand breaks (Hamperl and Cimprich, 2014;Sollier et al, 2014). To investigate if RNA-DNA hybrids might regulate ( pT371)TRF1 recruitment to APBs, we generated U2OS cell lines stably expressing the vector alone or Myc-tagged RNaseH1 (Fig.…”

“…Alternatively, it was possible that RNA-DNA hybrids at telomeres might exist transiently and that they might be sensitive to experimental conditions. To further investigate the role of RNA-DNA hybrids in regulating ( pT371)TRF1 association with APBs, we turned to campothecin (CPT), which has been reported to induce the formation of RNA-DNA hybrids (Sollier et al, 2014;Sordet et al, 2009). CPT is an inhibitor of topoisomerase I, which prevents the formation of RNA-DNA hybrids and resolves conflicts between transcription and replication (Hamperl and Cimprich, 2014;Tuduri et al, 2009).…”

Polymerase θ-helicase efficiently unwinds DNA and RNA-DNA hybrids

“…For example, R-loops have long been associated with replicative stress and genome instability, whereas more recent work indicates that RNA-DNA hybrids can also promote DNA repair by mechanisms that remain to be elucidated (23)(24)(25)(26)(27). Considering the importance of RNA-DNA structures in DNA repair and genome instability, we proceeded to examine whether Polθ-helicase unwinds RNA-DNA duplexes with similar efficiency.…”

Yra1-bound RNA–DNA hybrids cause orientation-independent transcription–replication collisions and telomere instability

“…Thus, RNA-DNA helicases are required for the replication of highly transcribed regions (Boubakri et al 2010), and R-loop accumulation impairs replication fork progression from bacteria to human cell lines (Wellinger et al 2006;Tuduri et al 2009;Gan et al 2011;CastellanoPozo et al 2012;Hamperl et al 2017). Alternatively, R loops may generate genomic instability if the displaced ssDNA is recognized and processed by flap endonucleases (Sollier et al 2014). Despite reports showing that R loops alter replication, causing genome instability, the mechanism is still unclear.…”