The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability

Abstract

Accurate DNA replication and DNA repair are crucial for the maintenance of genome stability, and it is generally accepted that failure of these processes is a major source of DNA damage in cells. Intriguingly, recent evidence suggests that DNA damage is more likely to occur at genomic loci with high transcriptional activity. Furthermore, loss of certain RNA processing factors in eukaryotic cells is associated with increased formation of co-transcriptional RNA:DNA hybrid structures known as R-loops, resulting in double-strand breaks (DSBs) and DNA damage. However, the molecular mechanisms by which R-loop structures ultimately lead to DNA breaks and genome instability is not well understood. In this review, we summarize the current knowledge about the formation, recognition and processing of RNA:DNA hybrids, and discuss possible mechanisms by which these structures contribute to DNA damage and genome instability in the cell.

Keywords: APOBEC family; ASF splicing factor; Activation-induced deaminase (AID); Double-strand breaks; G-quadruplex; Genome instability; R-loops; RNA processing factors; RNA:DNA helicases; RNase H; Senataxin; THO/TREX complex; THSC/TREX-2; Topoisomerase; Transcription–replication conflicts; mRNP biogenesis.

Figure 1. Schematic representation of an R-loop structure

The cartoon depicts the general structure of an R-loop. The nascent RNA strand (red) is synthesized by RNA polymerase (RNAP, red oval) and hybridizes with the complementary DNA template strand. The non-template strand is exposed as single-stranded DNA (ssDNA).

Figure 2. Formation of R-loops is facilitated by G-rich sequences and transcriptional supercoiling

A) R-loop formation is preferred for consecutive clusters of 3 or more G-residues on the non-template strand in the R-loop initiating zone (RIZ). G-rich sequences downstream in the elongation zone of the R-loop (REZ) facilitate extension of the RNA:DNA hybrid. B) Formation of RNA:DNA hybrids is also dependent on the stability of the resulting RNA:DNA hybrid and the exposed stretch of ssDNA. Clusters of G tracts on the non-template strand can fold into a stable G-quadruplex structure, which may help to stabilize the exposed ssDNA region of the R-loop. In addition, ssDNA binding proteins like RPA (yellow ovals) may associate and contribute to the stability of the vulnerable DNA strand. C) Positive and negative supercoils of the DNA double helix arise ahead of and behind the elongating RNAP, respectively. Negative supercoiling favors partial unwinding of the DNA double helix and may result in transient accumulation of ssDNA, thereby facilitating intrusion of the nascent RNA strand to hybridize with the complementary template strand.

Figure 3. Prevention and resolution of R-loop structures in the genome by multiple conserved mechanisms

A) Co-transcriptional assembly of RNP particles on the nascent RNA is a conserved strategy to prevent formation of R-loops from bacteria to metazoans. In bacteria, ribosomes associate cotranscriptionally with the nascent RNA and couple the two processes of transcription and translation. In this way, the RNA strand is prevented from forming an R-loop on the bacterial DNA template. In yeast and metazoan genomes, the double-helix is wrapped around nucleosome core particles (brown ovals) that may help to prevent invasion of the RNA strand after passage of RNAP. In addition, numerous RNA processing and splicing factors assemble with the RNA strand and prevent the accumulation of R-loops. Homologous subunits of the conserved protein complexes THO, TREX, and TREX-2/AMEX in yeast and metazoans are depicted in the same color. B) RNase H enzymes degrade the RNA moiety of the RNA:DNA hybrid. C) Specific helicases like Senataxin have been proposed to specifically unwind the RNA:DNA hybrid and allow re-annealing of the non-template strand to restore the DNA double helix.

Figure 4. Possible mechanisms for R-loop mediated formation of single-strand and double-strand breaks

A) R-loop mediated SSBs can be generated by a variety of factors/pathways. AID or other proteins of the APOBEC family were shown to deaminate cytosine residues preferentially on single-stranded DNA. The resulting uracil is recognized by proteins of the mismatch repair (MMR) or base excision repair (BER) machineries to create an abasic site, which is processed by apurinic-apyrimidinic endonucleases to generate a single-strand break. Alternatively, Topoisomerase 1 can be irreversibly trapped during its cleavage–ligation cycle, giving rise to a covalent Top1-DNA complex attached to the 5` end of the nicked DNA. This irreversible complex is likely to be processed into a small gap by specific endonucleases like Rad1/Rad10 (yeast) or Mus81/Mms4 (human), giving rise to a break or a small gap in the template or non-template strand. Finally, G-quadruplex or flap endonucleases may recognize a secondary DNA structure on the ssDNA or other structural features like the loop-duplex junction generating a single-strand break in the DNA. B) Possible mechanisms that induce R-loop mediated DSBs or convert the initiating SSB into a DSB. Green ovals represent DNA polymerases. Green hexagons indicate the replicative helicase. (1) A second SSB generated by one of the mechanisms described in A) may arise in proximity to the first SSB, which would result in a DSB (2). A replication fork may encounter the R-loop containing the SSB in co-directional (3) or head-on (6) orientation. If the fork can progress over the RNA:DNA hybrid (4, 7), separation of the parental strands over the SSB would result in a DNA lesion on the lagging (5) or leading strand (8), respectively. In the absence of a SSB at the R-loop structure, the stalled RNAP may still collide with the replication machinery in a co-directional (9) or head-on (10) orientation. Head-on collisions may be especially deleterious, leading to fork stalling and collapse, ultimately resulting in a DSB.