PARG inhibition reduces ssDNA levels and limits RPA loading upon replication fork collapse

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational modification crucial for DNA repair.
• PARG inhibition leads to sustained PARylation, presenting a potential anticancer strategy.
• The cellular consequences of persistent PARylation, especially during replication stress, are not fully understood.

Purpose of the Study:

• To investigate the impact of sustained PARylation on cellular responses to replication stress and DNA damage.
• To elucidate the role of PARylation in regulating RPA phosphorylation and chromatin association under fork-stalling conditions.

Main Methods:

• Utilized PARG inhibitors to induce sustained PARylation in cellular models.
• Assessed levels of phosphorylated and chromatin-bound RPA under various replication stress conditions.
• Investigated the involvement of PARP1, ATR-CHK1 signaling, and BRCA1 in the observed effects.
• Monitored single-stranded DNA (ssDNA) and RAD51 loading.

Main Results:

• Sustained PARylation significantly reduced phosphorylated and chromatin-bound RPA, particularly under conditions of replication fork collapse.
• This reduction was dependent on PARP1 activity and modulated by intact ATR-CHK1 signaling.
• Checkpoint inhibition rendered cells sensitive to PARG inhibitor-induced RPA loss.
• Reduced RPA phosphorylation and chromatin binding correlated with decreased exposed ssDNA, independent of BRCA1 and RAD51 loading.

Conclusions:

• Sustained PARylation creates a checkpoint-dependent fork-collapse state that limits RPA phosphorylation and ssDNA accumulation.
• This finding provides new insights into the cellular consequences of PARG inhibition and its potential as an anticancer therapy.