XPF interacts with TOP2B for R-loop processing and DNA looping on actively transcribed genes
Georgia Chatzinikolaou1, Kalliopi Stratigi1, Athanasios Siametis1,2
1Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, GR70013, Heraklion, Crete, Greece.
Abstract:
Co-transcriptional RNA-DNA hybrids can not only cause DNA damage threatening genome integrity but also regulate gene activity in a mechanism that remains unclear. Here, we show that the nucleotide excision repair factor XPF interacts with the insulator binding protein CTCF and the cohesin subunits SMC1A and SMC3, leading to R-loop-dependent DNA looping upon transcription activation. To facilitate R-loop processing, XPF interacts and recruits with TOP2B on active gene promoters, leading to double-strand break accumulation and the activation of a DNA damage response. Abrogation of TOP2B leads to the diminished recruitment of XPF, CTCF, and the cohesin subunits to promoters of actively transcribed genes and R-loops and the concurrent impairment of CTCF-mediated DNA looping. Together, our findings disclose an essential role for XPF with TOP2B and the CTCF/cohesin complex in R-loop processing for transcription activation with important ramifications for DNA repair-deficient syndromes associated with transcription-associated DNA damage.
Insights
This study reveals how RNA-DNA hybrids (R-loops) are processed during transcription. The nucleotide excision repair factor XPF, with TOP2B and CTCF/cohesin, facilitates R-loop resolution, impacting DNA looping and gene activation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Co-transcriptional RNA-DNA hybrids (R-loops) pose risks to genome integrity by causing DNA damage.
- The precise mechanisms by which R-loops regulate gene activity remain largely unknown.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying R-loop processing during transcription.
- To investigate the role of the nucleotide excision repair factor XPF in R-loop resolution and gene regulation.
Main Methods:
- Co-immunoprecipitation assays to identify protein interactions.
- Chromatin immunoprecipitation (ChIP) to assess protein recruitment to gene promoters.
- Analysis of DNA damage response markers and DNA looping.
Main Results:
- XPF interacts with CTCF and cohesin subunits (SMC1A, SMC3), mediating R-loop-dependent DNA looping during transcription activation.
- XPF recruits TOP2B to active gene promoters, promoting double-strand breaks and DNA damage response activation.
- TOP2B abrogation impairs the recruitment of XPF, CTCF, and cohesin, hindering DNA looping and R-loop processing.
Conclusions:
- XPF, TOP2B, and the CTCF/cohesin complex are crucial for processing R-loops during transcription activation.
- This pathway is vital for maintaining genome integrity and regulating gene expression.
- Findings have implications for understanding DNA repair-deficient syndromes linked to transcription-associated DNA damage.
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