Walking a tightrope: The complex balancing act of R-loops in genome stability

Abstract

Although transcription is an essential cellular process, it is paradoxically also a well-recognized cause of genomic instability. R-loops, non-B DNA structures formed when nascent RNA hybridizes to DNA to displace the non-template strand as single-stranded DNA (ssDNA), are partially responsible for this instability. Yet, recent work has begun to elucidate regulatory roles for R-loops in maintaining the genome. In this review, we discuss the cellular contexts in which R-loops contribute to genomic instability, particularly during DNA replication and double-strand break (DSB) repair. We also summarize the evidence that R-loops participate as an intermediate during repair and may influence pathway choice to preserve genomic integrity. Finally, we discuss the immunogenic potential of R-loops and highlight their links to disease should they become pathogenic.

Keywords: DNA damage; R-loop pathology; R-loops; RNA-DNA hybrid; double-strand break repair; genome stability; replication stress.

Figure 1.

Factors that Suppress and Resolve R-loops. Factors that prevent the formation of R-loops or play a role in their resolution as referenced throughout the text. The black line is parental DNA, blue lines are daughter DNA, and RNA is depicted by red lines. The stacked structure on the ssDNA strand of the R-loop represents a G-quadruplex.

Figure 2.

Factors Contributing to Replication Fork Stalling at R-loops. (A) Illustrates the two orientations in which a replication fork can encounter a R-loop, leading to a TRC. Head-on conflicts (HO, left) activate ATR and codirectional conflicts (CD, right) activate ATM. HO conflicts with R-loops are associated with greater genome instability. (B) Illustrates potential mechanisms that cause fork slowing in the HO (left) and CD (right) orientation. Flags represent epigenetic markers that promote chromatin compaction.

Figure 3.

Models for the Restart of Replication Forks Stalled at R-loops. (A-D) illustrate potential bypass mechanisms in the CD orientation. (A) The CMG helicase translocates over and bypasses a “naked” hybrid when the 5’-end of the RNA strand is annealed to the DNA. Once the hybrid is bypassed, the RNA strand can be extended by Pol α. (B) The CMG helicase unwinds a hybrid when the 5’-end of the RNA strand is exposed as a flap, allowing fork progression to continue. (C) CMG translocation over the hybrid would lead to its arrest at RNAP. Fork progression at this point would require CMG to bypass the stalled RNAP or for RNAP to be removed. (D) CMG unwinding of a hybrid bound to RNAP would allow RNAP to continue and the replication fork to progress behind transcription. (E-G) illustrate potential restart mechanisms in the HO orientation. (E) CMG bypass of RNAP would leave ssDNA exposed, allowing PrimPol recruitment. Primer synthesis by PrimPol allows the fork to continue, leaving behind ssDNA that can be replicated by a gap-filling mechanism once transcription is complete. (F) ssDNA resulting from bypass and repriming or a failure to reprime, and which persists behind the replication fork, could serve as a template for the formation of hybrids. Hybrids could form through de novo synthesis by RNAP (top) or through the association of nascent RNA resulting from transcription after the fork has passed (bottom). (G) Restart of a stalled fork initiated by SLX4 and MUS81-mediated fork cleavage is thought to relieve the torsional stress, resulting in resolution of the R-loop formed during a HO conflict. Religation of the fork by XRCC4/LIG4 and ELL-mediated restart of transcription allows both replication and transcription to continue. Prior to fork cleavage, RAD51 may promote fork reversal. RECQ1 is needed to reset the fork and RECQ5 promotes removal of RAD51.

Figure 4.

Potential Mechanisms for Resolution of R-loop Induced Transcription-Replication Conflicts. (A) In budding yeast, RNAP removal and proteasomal degradation can be initiated by activation of ATR and mediated by Paf1c and Ino80. RNAP phosphorylation also promotes its degradation, which is counteracted by the protein phosphatase PP1 and its regulatory subunit PNUTS and WDR82. (B) ATR activation at stalled forks is dependent upon MUS81-EME2-mediated fork cleavage, which is needed to generate ssDNA. Activation prevents additional and excessive fork cleavage by MUS81 and suppresses TRC formation. Other characterized functions of ATR may also promote fork recovery. (C) A fork stalled at an HO conflict can be rescued by a fork approaching the R-loop from the CD orientation. (D) Fork pausing at the 3’-end of a gene (highlighted in gray) located near an origin allows time for transcription to complete before the fork progresses into the gene. The converging fork and transcription lead to torsional stress that is relieved by TOP1. Pausing also activates ATR, which may reinforce the slowdown by inhibiting elongation. (E) Histone deacetylation suppresses R-loop formation in diverse ways, preventing genome instability. (F) SWI/SNF complexes including the subcomplex containing ARID1A act in a pathway with FANCD2 and SETX to suppress R-loop formation and TRCs. ARID1A recruits TOP2A to forks to relieve torsional stress.

Figure 5.

Potential Mechanisms of Replication-Independent R-loop-mediated DNA Damage. (A) The TC-NER nucleases XPG and XPF initiate R-loop processing by cleaving the 5’ or 3’-flap of the R-loop-template DNA junction, respectively. Cleavage may occur on either the template strand, excising the hybrid, or on the non-template strand, excising the displaced ssDNA to generate a single-strand break. Conversely, XPG and XPF may cleave opposing DNA strands of the R-loop, generating a DSB either 5’ or 3’ to the R-loop. (B) R-loop processing by several nucleases and TOP1-mediated DNA nicking in response to camptothecin generate two SSBs on opposing DNA strands, which mimic a DSB. (C) The displaced ssDNA of a transcription bubble is more accessible to damage from reactive oxygen species (ROS). ROS-generated SSBs in the displaced ssDNA promote unscheduled R-loops and G4s. (D) AID and APOBEC proteins can convert cytosine to uracil in the displaced ssDNA. Downstream processing of uracil results in an abasic site, which can cause SSBs. Additionally, the RNA editor ADAR has some activity towards the DNA strand of the hybrid. Base modifications are represented by stars.

Figure 6.

Unscheduled R-loops Interfere with DNA Repair. (A) Various helicases, including DDX5 and DHX9, act with BRCA1 to resolve hybrids that form when a DSB occurs in an actively transcribed locus. Loss of DDX5 or DHX9 results in a persistent R-loop at the break site, which inhibits MRN/CtIP-mediated end resection, leading to HR defects. (B) (i) In budding yeast, Sen1 and RNase H (RH) resolve hybrids. When both factors are lost, persistent R-loops result in a DSB forming downstream of the R-loop. While resection can occur normally on the hybrid-free end, the R-loop blocks MRN/CtIP activity, preventing resection and resulting in genomic instability and cell death. (ii) In wild-type cells, BRCA2 recognizes an R-loop near a DSB and recruits RNase H2 (RH2) and EXO1. EXO1 resects the DNA strand, while RNase H2 degrades the RNA, promoting RAD51 nucleofilament formation and proper HR. Upon loss of BRCA2 or RNase H2, the hybrid persists, blocking RAD51 loading and impairing HR. (C) In normal cells, BRCA1 associates with RNAP to promote transcription termination and resolve R-loops at termination sites. Upon DSB induction, BRCA1 dissociates from RNAP and traverses to the break site to promote repair. In Ewing’s Sarcoma patients, EWS-FLI prevents BRCA1 from dissociating with RNAP, even in the presence of DNA damage.

Figure 7.

Roles for R-loops and Hybrids in DNA Repair Pathways. (A) During NHEJ, KU recognizes the DSB break ends and recruits RNAPII. RNAPII may transcribe de novo RNA from the break site and recruit 53BP1. (B) MRN binds to DNA ends and melts the duplexed DNA. MRN also recruits RNAPII, which initiates de novo RNA synthesis, forming dilncRNAs. These RNAs are consecutively processed by Drosha and Dicer, generating DDRNAs (red dsRNAs). DDRNAs promote 53BP1 recruitment and aggregation, and NHEJ repair. (C) During HR, RNAPII synthesizes diRNA at the break site. diRNAs are processed by Dicer, and Ago2 interacts with the product. Ago2 binds RAD51 and localizes it to the break site, likely forming a hybrid between the processed diRNA and the resected end. (D) When a DSB occurs in an actively transcribed region, a complex of RAD51AP1 and UAF1 facilitates ssRNA strand invasion into the donor DNA (blue). This stimulates RAD51-mediated ssDNA invasion into the donor template. The resulting structure is called a DR-loop. After hybrid removal, HR proceeds as normal. (E) MRN/CtIP recognize DSB ends and recruit RNAPIII, which initiates de novo RNA synthesis. The DNA flap is excised by EXO1 and DNA2. RNAPIII-mediated hybrid formation promotes RAD51 loading. (F) An R-loop forms at a break site when a DSB occurs in an actively transcribed locus. RAD52 recognizes the R-loop and recruits XPG and BRCA1 to the break site. BRCA1 inhibits 53BP1 at the break, while XPG cleaves the junction 5’ to the hybrid, initiating resection and RPA loading. (G) When a SSB occurs near an R-loop that forms at a transcription termination site, BRCA1 recruits SETX, Dicer, and Ago2 to the SSB, which generates sdRNAs. These RNAs are recognized by the RAD52/PALB2 complex, which localize to the SSB site and promote repair. (H) Should a DSB occur in a transcribed region, RNA can mediate repair via at least two distinct mechanisms. (i) The reverse transcriptase TY generates cDNA from the RNA transcript. RAD52 then promotes invasion of the cDNA into the break ends, and downstream processing occurs to complete repair. This pathway is called cDNA-templated repair (c-TDR). (ii) During RNA-templated repair (R-TDR), RAD52 promotes nascent RNA invasion into the upstream break end, creating an RNA bridge. The DNA Polymerases Polθ or Polζ can synthesize DNA from the template, repairing the break.