The DNA-dependent protease AtWSS1A suppresses persistent double strand break formation during replication

Area of Science:

• Plant molecular biology
• DNA repair mechanisms
• Genome stability

Background:

• DNA-protein crosslinks (DPCs) pose a significant challenge to genome integrity.
• Protease WSS1A plays a role in DPC repair in plants.
• Replication fork stalling and collapse can lead to DNA double-strand breaks (DSBs).

Purpose of the Study:

• To investigate the function of WSS1A in DNA repair and genome stability in Arabidopsis.
• To determine the role of WSS1A in the context of other DNA repair pathways, including nonhomologous end joining (NHEJ) and the RTR complex.
• To elucidate the precise mechanism by which WSS1A contributes to preventing genome instability.

Main Methods:

• Arabidopsis thaliana genetic mutant analysis (wss1a mutants).
• CRISPR/Cas9-induced double-strand break (DSB) assays.
• Deep sequencing for DNA repair event analysis.
• Assessment of rDNA repeat copy number and chromosomal fragmentation.

Main Results:

• Loss of WSS1A leads to reduced 45S rDNA repeats and increased chromosomal fragmentation.
• WSS1A is essential for viability when DNA replication intermediate dissolution factors (RTR complex) are absent.
• Combined loss of WSS1A and NHEJ pathways (especially alternative NHEJ) exacerbates proliferation defects and chromosome fragmentation.
• CRISPR/Cas9-induced DSB repair analysis showed no significant change in repair quality, but a slight increase in quantity, suggesting WSS1A acts upstream of DSB formation.

Conclusions:

• WSS1A is critical for resolving complex DNA structures, likely DNA-protein crosslinks and replication intermediates.
• WSS1A functions in preventing the formation of persistent double-strand breaks (DSBs) that arise from unrepaired replication stress.
• WSS1A is essential for maintaining genome stability, particularly under conditions of impaired replication fork processing or DSB repair.