Toxic R-loops: Cause or consequence of replication stress?

doi:10.1016/j.dnarep.2021.103199

DNA Repair

Volume 107, November 2021, 103199

https://doi.org/10.1016/j.dnarep.2021.103199 Get rights and content

Abstract

Transcription-replication conflicts (TRCs) represent a potential source of endogenous replication stress (RS) and genomic instability in eukaryotic cells but the mechanisms that underlie this instability remain poorly understood. Part of the problem could come from non-B DNA structures called R-loops, which are formed of a RNA:DNA hybrid and a displaced ssDNA loop. In this review, we discuss different scenarios in which R-loops directly or indirectly interfere with DNA replication. We also present other types of TRCs that may not depend on R-loops to impede fork progression. Finally, we discuss alternative models in which toxic RNA:DNA hybrids form at stalled forks as a consequence - but not a cause - of replication stress and interfere with replication resumption.

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.

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Nonhomologous end-joining (NHEJ) factors act in replication-fork protection, restart, and repair. Here, we identified a mechanism related to RNA:DNA hybrids to establish the NHEJ factor Ku-mediated barrier to nascent strand degradation in fission yeast. RNase H activities promote nascent strand degradation and replication restart, with a prominent role of RNase H2 in processing RNA:DNA hybrids to overcome the Ku barrier to nascent strand degradation. RNase H2 cooperates with the MRN-Ctp1 axis to sustain cell resistance to replication stress in a Ku-dependent manner. Mechanistically, the need of RNaseH2 in nascent strand degradation requires the primase activity that allows establishing the Ku barrier to Exo1, whereas impairing Okazaki fragment maturation reinforces the Ku barrier. Finally, replication stress induces Ku foci in a primase-dependent manner and favors Ku binding to RNA:DNA hybrids. We propose a function for the RNA:DNA hybrid originating from Okazaki fragments in controlling the Ku barrier specifying nuclease requirement to engage fork resection.
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Histone chaperones are an important class of proteins that regulate chromatin accessibility for DNA-templated processes. Spt6 is a conserved histone chaperone and key regulator of transcription and chromatin structure. However, its functions outside of these roles have been little explored. In this work, we demonstrate a requirement for S. cerevisiae Spt6 in DNA replication and, more broadly, as a regulator of genome stability. Depletion or mutation of Spt6 impairs DNA replication in vivo. Additionally, spt6 mutants are sensitive to DNA replication stress-inducing agents. Interestingly, this sensitivity is independent of the association of Spt6 with RNA polymerase II (RNAPII), suggesting that spt6 mutants have a transcription-independent impairment of DNA replication. Specifically, genomic studies reveal that spt6 mutants have decreased loading of the MCM replicative helicase at replication origins, suggesting that Spt6 promotes origin licensing. Our results identify Spt6 as a regulator of genome stability, at least in part through a role in DNA replication.
RAD51 protects human cells from transcription-replication conflicts
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Citation 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.
Oncogene activation during tumorigenesis promotes DNA replication stress (RS), which subsequently drives the formation of cancer-associated chromosomal rearrangements. Many episodes of physiological RS likely arise due to conflicts between the DNA replication and transcription machineries operating simultaneously at the same loci. One role of the RAD51 recombinase in human cells is to protect replication forks undergoing RS. Here, we have identified a key role for RAD51 in preventing transcription-replication conflicts (TRCs) from triggering replication fork breakage. The genomic regions most affected by RAD51 deficiency are characterized by being replicated and transcribed in early S-phase and show significant overlap with loci prone to cancer-associated amplification. Consistent with a role for RAD51 in protecting against transcription-replication conflicts, many of the adverse effects of RAD51 depletion are ameliorated by inhibiting early S-phase transcription. We propose a model whereby RAD51 suppresses fork breakage and subsequent inadvertent amplification of genomic loci prone to experiencing TRCs.
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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.
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.
Biochemical and single-molecule techniques to study accessory helicase resolution of R-loop proteins at stalled replication forks
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Citation 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).
R-loop proteins present a stable and robust blockade to the progression of a DNA replication fork during S-phase. The consequences of this block can include mutagenesis and other irreversible chromosomal catastrophes, causing genomic instability and disease. As such, further investigation into the molecular mechanisms underlying R-loop protein resolution is warranted. The critical role of non-replicative accessory helicases in R-loop protein resolution has increasingly come into light in recent years. Such helicases include the Pif1-family, monomeric helicases that have been studied in many different contexts and that have been ascribed to a multitude of separable protective functions in the cell. In this chapter, we present protocols to study R-loop protein resolution by Pif1 helicase at stalled replication forks using purified proteins, both at the biochemical and single-molecule level. Our system uses recombinant proteins expressed in Saccharomyces cerevisiae but could apply to practically any organism of interest due to the high interspecies homology of the proteins involved in DNA replication. The methods we outline are extensible to many systems and should be applicable to studying R-loop clearance by any Superfamily (SF) 1B helicase. These techniques will further enable mechanistic research on these critical but understudied components of the genomic maintenance program.

View all citing articles on Scopus

^☆: This Special Issue is edited by P.A. Jeggo.

^☆☆: This article is part of the special issue Cutting Edge Perspectives in Genome Maintenance VIII.

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Toxic R-loops: Cause or consequence of replication stress?☆,☆☆

Abstract

Introduction

Section snippets

Direct effects: R-loops and RNA polymerases

How do eukaryotic cells avoid TRCs?

R-loops: cause or consequence of fork arrest?

Conclusion and perspectives

Declaration of Competing Interest

Acknowledgements

Cell

Mol. Cell

Mol. Cell

Mutat. Res.

Mol. Cell

Trends Genet.

Cell

Mol. Cell

Cell

Mol. Cell

Cell Rep.

Cell Rep.

J. Biol. Chem.

Mol. Cell

Cell

Mol. Cell

Mol. Cell

Cell

Mol. Cell

Cell Rep.

Cell

Cell

J. Biol. Chem.

Mol. Cell

Cell

Mol. Cell

Cell

Cell

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.