A specialized NHEJ pathway repairs DNA double-strand breaks in transcriptionally active genomic regions

• Applicant: Professor Dr. Markus Löbrich
• Subject Area: General Genetics and Functional Genome Biology ; Cell Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 553935806

The repair of DNA double-strand breaks (DSBs) is crucial for maintaining genomic stability. Non-homologous end-joining (NHEJ) and homologous recombination (HR) are the main DSB repair pathways which operate with different fidelity and have different cell cycle requirements. HR is a high-fidelity pathway since it uses the homologous sister chromatid as a template for repair but is therefore restricted to the S and G2 phases of the cell cycle. NHEJ, in contrast, has no intrinsic feature to restore sequence information that is lost at the break site and can operate in all cell cycle phases. A particular challenge for maintaining genomic stability is DSBs arising in transcribed regions since unfaithful repair will lead to the loss of gene function. Consequently, cells in S and G2 phase employ high-fidelity HR for repairing such breaks but how DSBs in transcribed genomic regions of G1-phase cells are repaired is unknown. We have previously established that NHEJ in G1 involves two components, a resection-independent component where break ends are rejoined with little or no processing of the ends and a resection-dependent component where substantial end-resection occurs prior to the rejoining step. How end-resection is compatible with a NHEJ process and how this affects the fidelity of NHEJ remained unclear. Further, it was unknown which DSBs undergo resection-dependent NHEJ and why. These how, which and why questions are addressed in the current proposal. We present preliminary evidence that a DNA fill-in reaction occurs at resected DSBs in G1-phase cells which reverts single-stranded DNA back to double-stranded DNA and enables the completion of NHEJ. We further provide evidence that DSBs arising in transcribed genomic regions of G1-phase cells undergo resection and fill-in synthesis and propose that this specialized NHEJ process serves to remove stalled RNA polymerases in the vicinity of the break site. We plan to characterize the fill-in process at resected DSBs and to assess the fidelity of this specialized NHEJ process. We also aim to show that the occupancy of RNA polymerases at DSB sites necessitates this NHEJ pathway and that the resection process removes stalled RNA polymerases to allow repair. We will employ immunofluorescence microscopy and various next generation sequencing techniques which can assess resection and fill-in synthesis at site-specific DSBs with nucleotide resolution. Collectively, our proposed studies will shed light on the question of how a NHEJ pathway repairs DSBs in transcribed genomic regions, a question of utmost importance for cells in the G0/G1 phase of the cell cycle including long-living post-mitotic cells such as neurons.

• DFG Programme: Research Grants