RAD54L coordinates the nucleolar DNA damage response to maintain rDNA stability
The nucleolus is organized around actively transcribed ribosomal RNA genes (rDNA), where high RNA polymerase I (Pol I) activity creates intrinsic susceptibility to replication stress and DNA damage. Here, we identify the DNA translocase RAD54L as a critical regulator of the nucleolar DNA damage response (nDDR) to rDNA double-strand breaks (DSBs) and replication stress. We show that RAD54L localizes to the nucleolus under basal conditions and is recruited to nucleolar caps following CRISPR-Cas9-induced rDNA-DSBs to promote repair. RAD54L loss results in persistent RAD51 foci, increased nucleolar γH2AX, and micronuclei formation, indicating defective resolution of rDNA lesions and genome instability. Under baseline conditions and replication stress induced by the Pol I transcription inhibitor CX-5461, RAD54L limits the accumulation of ssDNA and coordinates nDDR signaling. We further show that rDNA-DSBs induce RNA polymerase II-dependent RNA-DNA hybrids (R-loops) at intergenic rDNA regions, which facilitate nucleolar reorganization and cap formation and repair factor recruitment. Together, these findings establish RAD54L as a key regulator that coordinates replication stress response and rDNA repair, maintaining rDNA stability and genome integrity.
Authors
- Matthew J. Wakefield (ORCID: https://orcid.org/0000-0001-6624-4698)
- Jian Kang (ORCID: https://orcid.org/0000-0001-9998-4975)
- Y. C. Zhang
- Elaine Sanij (ORCID: https://orcid.org/0000-0002-2063-7813)
- Keefe T. Chan (ORCID: https://orcid.org/0000-0001-6114-3724)
- Kaylene J. Simpson (ORCID: https://orcid.org/0000-0001-9136-1781)
- Ruofei Liu (ORCID: https://orcid.org/0000-0003-1626-6041)
- Kezia Gitareja (ORCID: https://orcid.org/0000-0003-3397-7451)
- Karla J. Cowley (ORCID: https://orcid.org/0000-0001-9757-2627)
- Shannon Mendez
- Andrew J. Deans (ORCID: https://orcid.org/0000-0002-5271-4422)
- Jiachen Xuan
- Shalini Sundramurthi Chelliah (ORCID: https://orcid.org/0000-0003-3980-8560)
- Clare L. Scott (ORCID: https://orcid.org/0000-0002-3689-5956)
- Rhynelle Dmello
- Junqi Pan
- Carmelo Cerra
- Nyree Stojnic
Institutions
- Australian National University (AU)
- The University of Melbourne (AU)
- Walter and Eliza Hall Institute of Medical Research (AU)
- Australian Cancer Research Foundation (AU)
- Peter MacCallum Cancer Centre (AU)
- St Vincent's Hospital Melbourne (AU)
- Victorian Centre for Functional Genomics (AU)
- Monash University (AU)
- St Vincents Institute of Medical Research (AU)
Publication Details
- Journal
- The EMBO Journal
- Published
- 2026-07-23
- DOI
- https://doi.org/10.1038/s44318-026-00864-3
- Primary Topic
- DNA Repair Mechanisms
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Victorian Cancer Agency
- Australian Cancer Research Foundation
- Cancer Australia
- Tour de Cure
- National Health and Medical Research Council