Understanding The Roles Of SPIDR And PSO4in DNA Damage Response

To counteract DNA damage, eukaryotic cells have evolved the DNA damage response(DDR). DDR represents a complex network of multiple signaling pathways involving DNA repair, cell cycle checkpoints, transcriptional programs, and apoptosis, through which cells maintain genome stability following exposure to various endogenous or exogenous DNA-damaging agents.Of the different types of DNA damage that arise in cells, DNA double-strand break(DSB) is one of the most cytotoxic forms of DNA damage. Two major pathways for DSB repair have been defined:non-homologous end-joining (NHEJ) and homologous recombination (HR). HR repair plays a vital role in the maintenance of genomic integrity, and mutations in key components in the HR repair pathway are often associated with cancers and other human diseases. Bloom’s syndrome, characterized by growth deficiency, immunodeficiency, genome instability, and a high predisposition to cancer, is caused by mutation of the RecQ family gene BLM. BLM, the Bloom’s syndrome protein, is crucial for collaborating with HR machinery to promote efficient DNA repair and prevent DSB repair by less precise pathways. However, exactly how such coordination occurs in vivo is not clearly defined. Here, we identified a protein termed SPIDR (scaffolding protein involved in DNA repair) as the link between BLM and the HR machinery. SPIDR independently interacts with BLM and RAD51and promotes the formation of a BLM/RAD51-containing complex of biological importance. Consistent with its role as a scaffolding protein for the assembly of BLM and RAD51foci, depletion of SPIDR by RNA-mediated interference methods results in the high levels of sister-chromatid exchanges (SCE), the hallmark of Bloom’s syndrome cells. Moreover, SPIDR depletion leads to genome instability and causes hypersensitivity to DNA-damaging agents. We propose that, through providing a scaffold for the cooperation of BLM and RAD51in a multifunctional DNA-processing complex, SPIDR not only promotes the efficiency of HR, but also optimises the HR pathway and prevents unnecessary and potentially deleterious product in repair processes.The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase, a member of PIKK family, is a master checkpoint regulator of DDR. Upon DNA damage, the ATR-ATRIP (ATR-interacting protein) complex is recruited to DNA damage sites by RPA-ssDNA(replication protein A-single-stranded DNA), which is thought to play a crucial role in ATR activation. PSO4complex, containing PSO4/PRP19/SNEV, CDC5L, PLRG1, and SPF27, has a well-defined role in pre-mRNA splicing from yeast to humans. Recently, studies have described that the PSO4complex had a role in DDR, However, the specific role for the PSO4complex in DDR remains enigmatic. We showed that BCAS2subunit of the PSO4complex directly interacts with RPA1and is recruited to DNA damage sites in an RPA-dependent manner. Depletion of BCAS2or PSO4results in defective recruitment of ATRIP to DNA damage sites and compromises CHK1activation and RPA2phosphorylation. Moreover, we demonstrated that both the RPA1-binding ability of BCAS2and the E3ligase activity of PSO4are required for efficient accumulation of ATRIP at DNA damage sites and the subsequent CHK1activation and RPA2phosphorylation. Our results demonstrated that PSO4complex associates with RPA and regulates ATR-mediated cell-cycle checkpoint in response to genotoxic agents.