Chromatin modifications play an important role in transcription, DNA replication and repair. Nonetheless, whether histone modifications regulate replication stress responses remains obscure. Here, we show that RNF20 localizes to and promotes H2B monoubiquitination (H2Bub) at replicating sites. Knockdown of RNF20 leads to degradation of stalled forks by nucleolytic enzymes, which can be rescued by inhibition of MRE11/DNA2 and co-depletion of SMARCAL1/HLTF/ZRANB3 fork remodelers. RNF20 facilitates the loading of RAD51 and RAD51C at stalled fork sites and acts in the same pathway of RAD51/RAD51C-mediated fork protection and restart. Analyses with RING domain and phosphorylation-deficient mutants of RNF20 show that its catalytic activity and ATR-mediated phosphorylation are essential for its role in replication stress responses. Finally, treatment of RNF20-depleted cells with chromatin relaxing agents rescues fork protection and restart defects. Collectively, our study uncovers a role for RNF20-mediated H2Bub in regulating chromatin dynamics to safeguard replicating genomes.
Keywords: Fork Protection; Genome Stability; H2B Monoubiquitination; RNF20; Replication Stress.
© 2025. The Author(s).