Phosphorylation of replication protein A and its significance in the cellular response to DNA damage

Post-translational modification of proteins provides a cellular mechanism to switch on or off many diverse cellular processes. In response to slowed or stalled replication forks, which is defined as replication stress, phosphorylation enables rapid activation of proteins involved in DNA replication, repair, and cell cycle checkpoint signaling. One component of the replication stress response is Replication Protein A (RPA), a heterotrimeric protein with a middle subunit (RPA2) that becomes phosphorylated at multiple sites in a cell cycle-dependent as well as in a damage-dependent manner. Damage-induced hyperphosphorylation of RPA has been implicated to interfere with replication and to be involved in checkpoint signaling. This dissertation was undertaken to study the role of RPA phosphorylation in response to replication stress. Using Nijmegen breakage syndrome 1 (NBS1) cells stably transfected with vectors expressing phosphomutant forms of Nbs1, as well as HeLa cells transiently transfected with antibodies specific for the Nbs1 phosphorylation sites we found that hydroxyurea (HU)-induced RPA phosphorylation required ATR-dependent phosphorylation of NBS1. Interference with RPA hyperphosphorylation delayed HU-induced apoptosis.;We further demonstrated that RPA hyperphosphorylation was impaired in various head and neck squamous cell carcinoma (HNSCC) cell lines that displayed increased sensitivities to cisplatin and etoposide. Cisplatin-sensitive HNSCC that had been rendered more resistant to cisplatin by repeated exposure to cisplatin phosphorylated RPA2 in response to cisplatin and etoposide treatment. To investigate the consequences of impaired damage-induced RPA2 phosphorylation, we generated HNSCC cells stably expressing a phosphomutant RPA2 with ten phosphorylation sites mutated to alanines. The increased sensitivity to cisplatin treatment was paralleled by a defect in the recovery from cisplatin-induced replication slow-down and persistent replication stress. RPA2 mutant cells only displayed a minor increase in sensitivity to UV-C but showed decreased binding of RPA to ERCC1/XPF, an endonuclease implicated in crosslink repair and Nucleotide Excision Repair (NER). These data provide evidence for a critical role of damage-dependent RPA2 phosphorylation in the cellular response to replication stress, and suggest a requirement for phosphorylated RPA in crosslink repair that is distinct from its function in NER.