Proximity Ligation Assay for Detection of R-Loop Complexes upon DNA Damage

Alagia, Adele; Ketley, Ruth F; Gullerová, Monika

doi:10.1007/978-1-0716-2477-7_19

Cited by 9 publications

(3 citation statements)

References 20 publications

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“…Next, we investigated whether NSUN2 can be detected in proximity to R-loops in vivo . We performed a NSUN2-S9.6 optimised PLA assay 39 and detected an increased number of nuclear NSUN2-S9.6 foci in DNA damage conditions, which was reduced in the presence of transcription inhibitor DRB (Fig. 6c).…”

Section: Resultsmentioning

confidence: 89%

NSUN2 Facilitates DICER Cleavage of DNA Damage-Associated R-Loops to Promote Repair

Alagia,

Di Fazio,

Ajit

et al. 2024

Preprint

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DNA integrity is constantly challenged by both endogenous and exogenous damaging agents, resulting in various forms of damage. Failure to repair DNA accurately leads to genomic instability, a hallmark of cancer. Distinct pathways exist to repair different types of DNA damage. Double-strand breaks (DSBs) represent particularly severe form of damage, due to the physical separation of DNA strands. The repair of DSBs requires the activity of RNA Polymerase II (RNAPII) and the generation of Damage-associated transcripts (DARTs). Here we show that the RNA m5C-methyltransferase NSUN2 localizes to DSBs in a transcription-dependent manner, where it binds to and methylates DARTs. The depletion of NSUN2 results in an accumulation of nascent primary DARTs around DSBs. Furthermore, we detected an RNA-dependent interaction between NSUN2 and DICER, which was stimulated by DNA damage. NSUN2 activity promoted DICER cleavage of DARTs-associated R-loops, which is required for efficient DNA repair. We report a previously unrecognized role of the RNA m5C-methyltransferase NSUN2 within the RNA-dependent DNA damage response, highlighting its function as a DICER chaperone for the clearance of non-canonical substrates such as DARTs, thereby contributing to genomic integrity.

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Section: Resultsmentioning

confidence: 89%

NSUN2 Facilitates DICER Cleavage of DNA Damage-Associated R-Loops to Promote Repair

Alagia,

Di Fazio,

Ajit

et al. 2024

Preprint

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“…To further validate whether components of NuRD complex could interact with R-loops upon DNA damage in vivo , we employed optimized proximity ligation assay (PLA) that allows for detection of protein/R-loop complexes in vivo (Alagia et al , 2022). Using antibodies against endogenous GATAD2B or MBD3 and S9.6 we detected significant increase in PLA foci upon IR treatment.…”

Section: Resultsmentioning

confidence: 99%

GATAD2B containing NuRD complex drives R-loop dependent chromatin boundary formation at double strand breaks

Liu,

Ajit,

et al. 2024

Preprint

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Double-strand breaks (DSBs) are the most lethal form of DNA damage. Transcriptional activity at DSBs, as well as transcriptional repression around DSBs, are both required for efficient DNA repair. The chromatin landscape defines and coordinates these two opposing events. However, the regulation of the open and condensed chromatin architecture is still unclear. In this study, we show that the GATAD2B-NuRD complex associates with DSBs in a transcription- and R-loop-dependent manner, to promote histone deacetylation and chromatin condensation, creating a temporal boundary between open and closed chromatin. This boundary is necessary for correct DNA end resection termination. The lack of the GATAD2B-NuRD complex leads to chromatin hyper-relaxation and extended DNA end resection, resulting in HR repair failure. Our results suggest that the GATAD2B-NuRD complex is a key coordinator of the dynamic interplay between transcription and chromatin landscape and underscore its biological significance in the RNA-dependent DNA damage response.

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“…To test whether INTS6 is in close proximity to DNA:RNA hybrids in vivo, we performed a modified PLA using antibodies against INTS6 and S9.6 (recognizing DNA:RNA hybrids)(56) and observed a significant increased in PLA foci following IR ( Figure 1C , single antibodies were used as a negative control).…”

Section: Resultsmentioning

confidence: 99%

Tetrameric INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks

Long,

Ajit,

Sedova

et al. 2024

Preprint

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DNA double strand breaks (DSBs) represent a lethal form of DNA damage that can trigger cell death and initiate oncogenesis. The activity of RNA polymerase II (RNAPII) at the break site is required for efficient DSB repair. However, the regulatory mechanisms governing the transcription cycle at DSBs are not well understood. Here, we show that Integrator complex subunit 6 (INTS6) associates with the trimeric SOSS1 (comprising INTS3, INIP, and hSSB1) to form a tetrameric SOSS1 complex following DNA damage. INTS6 binds to DNA:RNA hybrids and plays a crucial role in Protein Phosphatase 2 (PP2A) recruitment to DSBs, facilitating the dephosphorylation of RNAPII. Furthermore, INTS6 prevents the accumulation of damage-induced RNA transcripts (DARTs) and the stabilization of DNA:RNA hybrids at DSB sites. INTS6 interacts with, and promotes the recruitment of Senataxin (SETX) to DSBs, facilitating the resolution of DNA:RNA hybrids/R-loops. Our results underscore the significance of the SOSS1 complex in the autoregulation of DNA:RNA dynamics and the promotion of efficient DNA repair.

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Proximity Ligation Assay for Detection of R-Loop Complexes upon DNA Damage

Cited by 9 publications

References 20 publications

NSUN2 Facilitates DICER Cleavage of DNA Damage-Associated R-Loops to Promote Repair

NSUN2 Facilitates DICER Cleavage of DNA Damage-Associated R-Loops to Promote Repair

GATAD2B containing NuRD complex drives R-loop dependent chromatin boundary formation at double strand breaks

Tetrameric INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks

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