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

Kemiha, Samira; Poli, Jérôme; Lin, Yea-Lih; Lengronne, Armelle; Pasero, Philippe

doi:10.1016/j.dnarep.2021.103199

Cited by 19 publications

(12 citation statements)

References 133 publications

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Section: Resultsmentioning

confidence: 99%

“…They have both regulatory and toxic effects on cells and have been the focus of many recent studies. (Bayona-Feliu and Aguilera, 2021;Castillo-Guzman and Chédin, 2021;Crossley et al, 2019;Kemiha et al, 2021) If increased RNA-DNA hybrid formation in spt6 mutants contributed to genome instability phenotypes, we would expect that altering RNA-DNA hybrid levels might enhance or suppress spt6 mutant phenotypes. To test this, we manipulated the levels of the RNA-DNA hybrid specific endoribonuclease RNaseH in our mutants.…”

Section: R-loops Are Not a Major Contributor To Spt6-mediated Genome ...mentioning

confidence: 99%

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The conserved histone chaperone Spt6 facilitates DNA replication and mediates genome instability

Miller

Winston

2022

Preprint

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SUMMARYHistone 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 role for S. cerevisiae Spt6 in DNA replication and more broadly as a regulator of genome stability. Spt6 binds the replication machinery and depletion or mutation of Spt6 impairs DNA replication in vivo. Additionally, spt6 mutants are sensitive to DNA replication stress inducing agents, with increased sensitivity when combined with loss of DNA replication associated factors. Furthermore, spt6 mutants have elevated levels of DNA double strand breaks and recombination. These effects appear to be independent of R-loops, which are not elevated in spt6 mutants. Our results identify Spt6 as a regulator of genome stability, at least in part through a role in DNA replication.

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Section: Resultsmentioning

confidence: 99%

Section: R-loops Are Not a Major Contributor To Spt6-mediated Genome ...mentioning

confidence: 99%

The conserved histone chaperone Spt6 facilitates DNA replication and mediates genome instability

Miller

Winston

2022

Preprint

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“…Despite increasing evidence that transcription is a primary source of replication impairment and genome instability 2 , much remains to be understood about the structures that are formed when these two processes interfere with each other, whether R-loops are the cause or consequence of RS and DNA damage 3 , and how the multiple factors and pathways that prevent or resolve them gain access to specific structures or genomic loci during the cell cycle 64 . Addressing these issues is crucial to harnessing RS and identifying cancer cell vulnerabilities.…”

Section: Discussionmentioning

confidence: 99%

“…Although the mechanisms that protect cells against exogenous sources of RS have been extensively characterized, how endogenous RS is dealt with by the cell is still elusive. Even in the absence of external assaults, replication forks can be hindered by lesions generated as a consequence of cellular metabolism and non-canonical DNA structures such as G-quadruplexes, hairpins, RNA:DNA hybrids or R-loops (three-stranded structures composed of an RNA − DNA hybrid and a displaced single-stranded DNA) that may be formed as a result of TRCs 2,3 . To allow faithful genome replication in the face of RS and avoid mitotic division with under-replicated DNA, cells possess protective mechanisms that stabilize, remodel or restart stalled replication forks, while delaying cell cycle progression 4 .…”

mentioning

confidence: 99%

FANCD2 promotes mitotic rescue from transcription-mediated replication stress in SETX-deficient cancer cells

et al. 2022

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Replication stress (RS) is a leading cause of genome instability and cancer development. A substantial source of endogenous RS originates from the encounter between the transcription and replication machineries operating on the same DNA template. This occurs predominantly under specific contexts, such as oncogene activation, metabolic stress, or a deficiency in proteins that specifically act to prevent or resolve those transcription-replication conflicts (TRCs). One such protein is Senataxin (SETX), an RNA:DNA helicase involved in resolution of TRCs and R-loops. Here we identify a synthetic lethal interaction between SETX and proteins of the Fanconi anemia (FA) pathway. Depletion of SETX induces spontaneous under-replication and chromosome fragility due to active transcription and R-loops that persist in mitosis. These fragile loci are targeted by the Fanconi anemia protein, FANCD2, to facilitate the resolution of under-replicated DNA, thus preventing chromosome mis-segregation and allowing cells to proliferate. Mechanistically, we show that FANCD2 promotes mitotic DNA synthesis that is dependent on XPF and MUS81 endonucleases. Importantly, co-depleting FANCD2 together with SETX impairs cancer cell proliferation, without significantly affecting non-cancerous cells. Therefore, we uncovered a synthetic lethality between SETX and FA proteins for tolerance of transcription-mediated RS that may be exploited for cancer therapy.

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“…Another consideration is that RNA:DNA hybrids appear to contribute to the TRCs observed in the trf4Δ mutant as we observed a suppression of TRCs upon overexpression of RNase H1, indicating that degradation of hybrids could allow for TRC resolution. It has been recently proposed that hybrids form at stalled forks and interfere with fork restart [75, 76]. It is possible that low levels of RPA due to sequestration by excess RNAs in the absence of TRAMP4 favors formation of hybrids behind the fork, inhibiting TRC resolution (Fig.…”

Section: Discussionmentioning

confidence: 99%

THO and TRAMP complexes prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions

Brown

Fair

et al. 2021

Preprint

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Expansion of structure-forming CAG/CTG repetitive sequences is the cause of several neurodegenerative disorders and deletion of repeats is a potential therapeutic strategy. Transcription-associated mechanisms are known to cause CAG repeat instability. In this study, we discovered that Thp2, an RNA export factor and member of the THO complex, and Trf4, a key component of the TRAMP complex involved in nuclear RNA degradation, are necessary to prevent CAG fragility and repeat contractions in a S. cerevisiae model system. Depletion of both Thp2 and Trf4 proteins causes a highly synergistic increase in CAG repeat fragility, indicating a complementary role of the THO and TRAMP complexes in preventing genome instability. Loss of either Thp2 or Trf4 causes an increase in RNA polymerase stalling at the CAG repeats and genome-wide transcription-replication conflicts (TRCs), implicating impairment of transcription elongation as a cause of CAG fragility and instability in their absence. Analysis of the effect of RNase H1 overexpression on CAG fragility and TRCs suggests that co-transcriptional R-loops are the main cause of CAG fragility in the thp2∆ mutants. In contrast, CAG fragility and TRCs in the trf4∆ mutant can be compensated for by RPA overexpression, suggesting that excess unprocessed RNA in TRAMP4 mutants leads to reduced RPA availability and high levels of TRCs. Our results show the importance of RNA surveillance pathways in preventing RNAPII stalling, TRCs, and DNA breaks, and show that RNA export and RNA decay factors work collaboratively to maintain genome stability.

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

Cited by 19 publications

References 133 publications

The conserved histone chaperone Spt6 facilitates DNA replication and mediates genome instability

The conserved histone chaperone Spt6 facilitates DNA replication and mediates genome instability

FANCD2 promotes mitotic rescue from transcription-mediated replication stress in SETX-deficient cancer cells

THO and TRAMP complexes prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions

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