RAD54L coordinates the nucleolar DNA damage response to maintain rDNA stability

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• The nucleolus, rich in ribosomal RNA genes (rDNA), is prone to replication stress and DNA damage due to high RNA polymerase I (Pol I) activity.
• The DNA damage response within the nucleolus (nucleolar DNA damage response or nDDR) is critical for maintaining genomic integrity.

Purpose of the Study:

• To identify key regulators of the nDDR, particularly in response to rDNA double-strand breaks (DSBs) and replication stress.
• To elucidate the role of RAD54L in nucleolar DNA repair and genome stability.

Main Methods:

• CRISPR-Cas9-induced rDNA-DSBs to study DNA repair.
• Replication stress induction using the Pol I inhibitor CX-5461.
• Analysis of DNA repair foci (RAD51, γH2AX), ssDNA accumulation, and micronuclei formation.
• Investigation of RNA polymerase II-dependent RNA-DNA hybrids (R-loops).

Main Results:

• RAD54L is identified as a critical regulator of the nDDR, localizing to the nucleolus and being recruited to rDNA-DSBs.
• RAD54L deficiency leads to unresolved rDNA lesions, increased γH2AX, RAD51 foci, and micronuclei, indicating genome instability.
• RAD54L limits ssDNA accumulation and coordinates nDDR signaling during replication stress.
• rDNA-DSBs induce R-loops that facilitate nucleolar reorganization and repair factor recruitment.

Conclusions:

• RAD54L is essential for coordinating the replication stress response and rDNA repair, thereby maintaining rDNA stability.
• The study establishes RAD54L as a key player in safeguarding genome integrity within the nucleolus.