RAD54L coordinates the nucleolar DNA damage response to maintain rDNA stability

  • EMBO J. 2026 Sep;45(17):6299-6319. doi: 10.1038/s44318-026-00864-3.
Ruofei Liu  #  1 ,  Jiachen Xuan  #  2  3 ,  Carmelo Cerra  1 ,  Nyree Stojnic  1 ,  Junqi Pan  4 ,  Shalini S Chelliah  1  5 ,  Shannon Mendez  1 ,  Rhynelle Dmello  1 ,  Karla J Cowley  6  7 ,  Yangyi Zhang  1 ,  Kezia Gitareja  1  8 ,  Matthew Wakefield  4  9 ,  Andrew Deans  1  8 ,  Clare L Scott  4  9 ,  Keefe T Chan  2 ,  Kaylene J Simpson  2  6  7 ,  Jian Kang  1  8 ,  Elaine Sanij  10  11  12  13
Affiliations
  • 1. St Vincent's Institute of Medical Research, Melbourne, VIC, Australia.
  • 2. Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC, Australia.
  • 3. The ACRF Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia.
  • 4. The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia.
  • 5. Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia.
  • 6. Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.
  • 7. Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.
  • 8. Department of Medicine- St Vincent's Hospital, University of Melbourne, Melbourne, VIC, Australia.
  • 9. Department of Obstetrics, Gynaecology and Newborn Health, University of Melbourne, Melbourne, VIC, Australia.
  • 10. St Vincent's Institute of Medical Research, Melbourne, VIC, Australia. [email protected].
  • 11. Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia. [email protected].
  • 12. Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia. [email protected].
  • 13. Department of Medicine- St Vincent's Hospital, University of Melbourne, Melbourne, VIC, Australia. [email protected].
  • # Contributed equally.
PMID: 42493648 DOI: 10.1038/s44318-026-00864-3
Abstract

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.

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