Consequences and Resolution of Transcription–Replication Conflicts

Lalonde, Maxime; Trauner, Michael; Werner, Marcel; Hamperl, Stephan

doi:10.3390/life11070637

Cited by 27 publications

(17 citation statements)

References 167 publications

(236 reference statements)

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“…One possible explanation for DSBs at the expanded CAG tract is TRCs due to a stalled RNAPII complex interfering with DNA replication [ 62 ]. To determine whether TRCs were occurring in thp2Δ , trf4Δ , or rnh1Δrnh201Δ mutants, we employed a proximity ligation assay (PLA) using antibodies against PCNA to detect the replisome and RNAPII to detect the transcription machinery.…”

Section: Resultsmentioning

confidence: 99%

The RNA export and RNA decay complexes THO and TRAMP prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions

Brown

Fair

et al. 2022

PLoS Biol

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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 (suppressors of transcriptional defects of hpr1Δ by overexpression) complex, and Trf4, a key component of the TRAMP (Trf4/5-Air1/2-Mtr4 polyadenylation) complex involved in nuclear RNA polyadenylation and degradation, are necessary to prevent CAG fragility and repeat contractions in a Saccharomyces 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 other genomic loci, as well as genome-wide transcription-replication conflicts (TRCs), implicating TRCs as a cause of CAG fragility and instability in their absence. Analysis of the effect of RNase H1 overexpression on CAG fragility, RNAPII stalling, and TRCs suggests that RNAPII stalling with associated R-loops are the main cause of CAG fragility in the thp2Δ mutants. In contrast, CAG fragility and TRCs in the trf4Δ 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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Section: Resultsmentioning

confidence: 99%

The RNA export and RNA decay complexes THO and TRAMP prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions

Brown

Fair

et al. 2022

PLoS Biol

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show abstract

“…One possible explanation for DSBs at the expanded CAG tract is TRCs due to a stalled RNA polymerase II complex (RNAPII) interfering with DNA replication [57]. To determine whether TRCs were occurring in thp2Δ, trf4Δ , or rnh1Δrnh201Δ mutants, we employed a proximity ligation assay (PLA) using antibodies against PCNA to detect the replisome, and RNAPII to detect the transcription machinery.…”

Section: Resultsmentioning

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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“…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%

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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Consequences and Resolution of Transcription–Replication Conflicts

Cited by 27 publications

References 167 publications

The RNA export and RNA decay complexes THO and TRAMP prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions

The RNA export and RNA decay complexes THO and TRAMP prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions

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

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

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