Toxic R-loops: Cause or consequence of replication stress?☆,☆☆
Introduction
R-loops are stable structures that form during transcription when the nascent RNA reanneals with the template DNA, generating an RNA:DNA hybrid and a displaced single-stranded DNA (ssDNA) loop [1]. Different techniques have been developed to analyze R-loops, from individual loci to the whole genome level [2]. These structures differ by their length, sequence and genomic context. They are very abundant, covering up to 5% of mammalian genomes [3,4]. R-loops play multiple physiological roles such as the regulation of immunoglobulin (Ig) class-switch recombination, CRISPR-Cas9 activity, DSB repair, initiation of mitochondrial DNA replication, chromatin patterning and gene regulation, including the protection of CpG islands against DNA methylation and the regulation of transcription termination [1,[5], [6], [7], [8]]. R-loops represent also a potential source of genomic instability, presumably by increasing transcription-replication conflicts. Indeed, the replication and transcription machineries translocate along the same DNA template and may interfere with each other. TRCs can either occur in a head-on or in a codirectional manner with different outcomes, head-on conflicts being more deleterious than codirectional ones [9,10]. Head-on conflicts have been associated with replication fork slowdown and with increased transcription-associated recombination [[11], [12], [13], [14], [15]]. Both events are suppressed by the overexpression of RNase H [[16], [17], [18]], a nuclease degrading the RNA moiety of RNA:DNA hybrids [19]. It is therefore generally believed that cotranscriptional R-loops interfere with DNA replication, but the mechanism(s) involved remain poorly understood at the molecular level.
In this review, we discuss several non-mutually exclusive processes by which transcription may interfere with DNA replication in eukaryotic cells, involving or not R-loops. We do not elaborate on the many factors that contribute to the formation or to the elimination of R-loops (Fig. 1) nor on the human pathologies associated with deregulated R-loop homeostasis as these topics have been extensively covered in recent reviews [1,5,6,20]. We rather discuss the multiple strategies used by eukaryotic cells to restrain transcription-replication conflicts in normal growth conditions. We also present alternative models in which RNA:DNA hybrids form at stalled forks as a consequence of replication arrest, and interfere with fork repair mechanisms, as recently reported for double-strand DNA break (DSB) repair.
Section snippets
Direct effects: R-loops and RNA polymerases
It is now well established that active and backtracked RNA polymerases can directly impede the progression of replication forks in bacteria [[21], [22], [23], [24], [25], [26]]. However, the impact of transcription on eukaryotic DNA replication is less well understood. Transcription interferes with the licensing of replication origins by displacing pre-replicative complexes or by altering their chromatin environment [[27], [28], [29], [30], [31], [32], [33], [34]]. Transcription can also impede
How do eukaryotic cells avoid TRCs?
The data discussed above argues against the view that R-loops represent direct obstacles for DNA replication and call for a reconsideration of the real impact of transcription-replication conflicts on the stability of the genome. Indeed, TRCs are usually studied in pathological conditions, under which replication is challenged by chemical inhibitors or by the absence of key regulators of R-loop formation. For instance, conflicts between replication and transcription have been implicated in
R-loops: cause or consequence of fork arrest?
The results discussed above seem to argue against a model in which R-loops directly block the progression of DNA replication forks, at least in normal growth conditions. Yet, most of these studies also show that the overexpression of RNase H largely rescues the slow replication phenotype associated with increased R-loops. This apparent discrepancy could be explained by an alternative model in which toxic R-loops are not a cause, but rather a consequence of replication fork arrest (Fig. 4).
Conclusion and perspectives
In conclusion, the mechanisms by which RNA:DNA hybrids interfere with DNA replication are more varied than initially thought and are probably not mutually exclusive. Although they could impede fork progression by acting directly or indirectly as roadblocks, they could also interfere with the restart of arrested forks, as they impede DSB repair. In cells that are unable to process R-loops, these hybrids could also delay the repair of replication-borne DSBs, inducing a persistent DNA damage
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
We thank Benjamin Pardo and Hervé Técher for helpful comments on the manuscript. SK thanks the Ministère de l'enseignement supérieur, de la recherche et de l'innovation (MESRI) for fellowship. Work in the Pasero lab is supported by grants from the Agence Nationale pour la Recherche (ANR), Institut National du Cancer (INCa), the Ligue Nationale Contre le Cancer (équipe labellisée), and the Fondation MSDAvenir.
References (151)
- et al.
R Loops: from physiological to pathological roles
Cell
(2019) - et al.
R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters
Mol. Cell
(2012) Prevalent, dynamic, and conserved R-loop structures associate with specific epigenomic signatures in mammals
Mol. Cell
(2016)- et al.
The dark side of RNA:DNA hybrids
Mutat. Res.
(2020) - et al.
R-loops as cellular regulators and genomic threats
Mol. Cell
(2019) Nascent connections: R-loops and chromatin patterning
Trends Genet.
(2016)- et al.
Conflict resolution in the genome: how transcription and replication make it work
Cell
(2016) - et al.
Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination
Mol. Cell
(2003) - et al.
Linking RNA polymerase backtracking to genome instability in E. coli
Cell
(2011) Post-licensing specification of eukaryotic replication origins by facilitated Mcm2-7 sliding along DNA
Mol. Cell
(2015)
INO80C remodeler maintains genomic stability by preventing promiscuous transcription at replication origins
Cell Rep.
Persistence of RNA transcription during DNA replication delays duplication of transcription start sites until G2/M
Cell Rep.
Perturbation of the activity of replication origin by meiosis-specific transcription
J. Biol. Chem.
Highly transcribed RNA polymerase II genes are impediments to replication fork progression in Saccharomyces cerevisiae
Mol. Cell
Senataxin associates with replication forks to protect fork integrity across RNA-polymerase-II-transcribed genes
Cell
Epigenetic instability due to defective replication of structured DNA
Mol. Cell
Transcription-coupled nucleotide excision repair factors promote R-loop-induced genome instability
Mol. Cell
Convergent transcription at intragenic super-enhancers targets AID-initiated genomic instability
Cell
R loops are linked to histone h3 s10 phosphorylation and chromatin condensation
Mol. Cell
Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts
Cell Rep.
Genome-organizing factors Top2 and Hmo1 prevent chromosome fragility at sites of S phase transcription
Cell
Chapter Four - coordinating cell cycle remodeling with transcriptional activation at the drosophila MBT
The replication checkpoint protects fork stability by releasing transcribed genes from nuclear pores
Cell
Emerging roles for R-loop structures in the management of topological stress
J. Biol. Chem.
Collisions between replication and transcription complexes cause common fragile site instability at the longest human genes
Mol. Cell
Identification of early replicating fragile sites that contribute to genome instability
Cell
A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability
Mol. Cell
A replication fork barrier at the 3’ end of yeast ribosomal RNA genes
Cell
Termination of mammalian rDNA replication: polar arrest of replication fork movement by transcription termination factor TTF-I
Cell
Mrc1 and Tof1 promote replication fork progression and recovery independently of Rad53
Mol. Cell
Best practices for the visualization, mapping, and manipulation of R-loops
EMBO J.
R-loops as Janus-faced modulators of DNA repair
Nat. Cell Biol.
Transcription-mediated replication hindrance: a major driver of genome instability
Genes Dev.
Replication fork progression is impaired by transcription in hyperrecombinant yeast cells lacking a functional THO complex
Mol. Cell. Biol.
Transcription-associated recombination is dependent on replication in mammalian cells
Mol. Cell. Biol.
Transcription-replication conflict orientation modulates R-loop levels and activates distinct DNA damage responses
Cell
Impairment of replication fork progression mediates RNA polII transcription-associated recombination
EMBO J.
Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription
Nat. Cell Biol.
Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites
Nat. Commun.
UAP56/DDX39B is a major cotranscriptional RNA-DNA helicase that unwinds harmful R loops genome-wide
Genes Dev.
Overexpression of RNase H partially complements the growth defect of an Escherichia coli delta topA mutant: R-loop formation is a major problem in the absence of DNA topoisomerase I
Proc. Natl. Acad. Sci. U. S. A.
Out of balance: R-loops in human disease
PLoS Genet.
Direct restart of a replication fork stalled by a head-on RNA polymerase
Science
Co-directional replication-transcription conflicts lead to replication restart
Nature
Head-on collision between a DNA replication apparatus and RNA polymerase transcription complex
Science
R-loop-mediated genomic instability is caused by impairment of replication fork progression
Genes Dev.
Replisome bypass of transcription complexes and R-loops
Nucleic Acids Res.
Pervasive transcription fine-tunes replication origin activity
Elife
Noncoding transcription influences the replication initiation program through chromatin regulation
Genome Res.
Transcription-mediated organization of the replication initiation program across large genes sets common fragile sites genome-wide
Nat. Commun.
Cited by (11)
Regulation and function of R-loops at repetitive elements
2023, BiochimieRAD51 protects human cells from transcription-replication conflicts
2022, Molecular CellCitation Excerpt :There is a strong association between an accumulation of R-loops and both TRCs and transcription-coupled genome instability (Santos-Pereira and Aguilera, 2015; Hamperl et al., 2017). These R-loops perturb replication fork progression (Brüning and Marians, 2020; Kemiha et al., 2021) and promote fork reversal (Chappidi et al., 2020; Matos et al., 2020). In the current study, we found that TRCs arising in absence of RAD51-mediated fork protection induce atypical MiDAS that is associated with R-loops.
Walking a tightrope: The complex balancing act of R-loops in genome stability
2022, Molecular CellCitation Excerpt :Finally, the remodelers INO80 (Poli et al., 2016; Prendergast et al., 2020) and ATRX have been implicated in TRC resolution and/or R-loop suppression (Nguyen et al., 2017a; Yan et al., 2022). An emerging question in the field is whether fork slowing at an R-loop is actually problematic for the cell or whether post-replicative hybrid formation is an additional culprit (Kemiha et al., 2021). Although the idea that forks slow upon approaching an R-loop is certainly the dominant model, it is important to better establish whether slowing and fork reversal actually occurs in front of the R-loop.
Biochemical and single-molecule techniques to study accessory helicase resolution of R-loop proteins at stalled replication forks
2022, Methods in EnzymologyCitation Excerpt :These blocks can include RNA-DNA hybrids with displaced single-stranded (ss) DNA (a.k.a. R-loops), DNA secondary structures such as G-quadruplexes, interstrand crosslinks from chemical agents and chemotherapeutics, actively transcribing RNA polymerases, and other protein complexes (Brüning, Howard, & McGlynn, 2014; Gómez-González & Aguilera, 2019; Perera, Behrmann, Hoang, Griffin, & Trakselis, 2019; Rickman & Smogorzewska, 2019; Semlow & Walter, 2021). Of particular interest are R-loop protein blocks such as active transcription complexes, as they are endogenous to the cell yet present robust barriers to replication that are often directly mutagenetic (Azvolinsky, Giresi, Lieb, & Zakian, 2009; Crossley, Bocek, & Cimprich, 2019; García-Muse & Aguilera, 2019; Helmrich, Ballarino, & Tora, 2011; Kemiha, Poli, Lin, Lengronne, & Pasero, 2021; Lalonde, Trauner, Werner, & Hamperl, 2021; Saponaro, 2022; St Germain, Zhao, & Barlow, 2021). Replication fork pausing at R-loops can arise from topological stress from positive supercoiling ahead of the fork, or from a simple physical blockade of replisome translocation along its template (Shyian & Shore, 2021).
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This Special Issue is edited by P.A. Jeggo.
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This article is part of the special issue Cutting Edge Perspectives in Genome Maintenance VIII.