DNA polymerase delta interacting protein 3 facilitates the activation and maintenance of DNA damage checkpoint in response to replication stress

Background:Replication stress response is crucial for the maintenance of a stable ge-nome.POLDIP3(DNA polymerase delta interacting protein 3)was initially identified as one of the DNA polymeraseδ(Polδ)interacting proteins almost 20 years ago.Using a variety of in vitro biochemical assays,we previously established that POLDIP3 is a key regulator of the enzymatic activity of Polδ.However,the in vivo function of POLDIP3 in DNA replication and DNA damage response has been elusive.Methods:We first generated POLDIP3 knockout(KO)cells using the CRISPR/Cas9 technology.We then investigated its biological functions in vivo using a variety of biochemical and cell biology assays.Results:We showed that although the POLDIP3-KO cells manifest no pronounced defect in global DNA synthesis under nonstress conditions,they are sensitive to a va-riety of replication fork blockers.Intriguingly,we found that POLDIP3 plays a crucial role in the activation and maintenance of the DNA damage checkpoint in response to exogenous as well as endogenous replication stress.Conclusion:Our results indicate that when the DNA replication fork is blocked,POLDIP3 can be recruited to the stalled replication fork and functions to bridge the early DNA damage checkpoint response and the later replication fork repair/restart.

Hsp90-associated DNA replication checkpoint protein and proteasome-subunit components are involved in the age-related macular degeneration

Background:Age-related macular degeneration(AMD)is the leading cause of vision loss worldwide.However,the mechanisms involved in the development and progression of AMD are poorly delineated.We aimed to explore the critical genes involved in the progression of AMD.Methods:The differentially expressed genes(DEGs)in AMD retinal pigment epithelial(RPE)/choroid tissues were identified using the microarray datasets GSE99248 and GSE125564,which were downloaded from the gene expression omnibus database.The overlapping DEGs from the two datasets were screened to identify DEG-related biological pathways using gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses.The hub genes were identified from these DEGs through protein-protein interaction network analyses.The expression levels of hub genes were evaluated by quantitative real-time polymerase chain reaction following the induction of senescence in ARPE-19 with FK866.Following the identification of AMD-related key genes,the potential small molecule compounds targeting the key genes were predicted by PharmacoDB.Finally,a microRNA-gene interaction network was constructed.Results:Microarray analyses identified 174 DEGs in the AMD RPE compared to the healthy RPE samples.These DEGs were primarily enriched in the pathways involved in the regulation of DNA replication,cell cycle,and proteasome-mediated protein polyubiquitination.Among the top ten hub genes,HSP90AA1,CHEK1,PSMA4,PSMD4,and PSMD8 were upregulated in the senescent ARPE-19 cells.Additionally,the drugs targeting HSP90AA1,CHEK1,and PSMA4 were identified.We hypothesize that Hsa-miR-16-5p might target four out of the five key DEGs in the AMD RPE.Conclusions:Based on our findings,HSP90AA1 is likely to be a central gene controlling the DNA replication and proteasome-mediated polyubiquitination during the RPE senescence observed in the progression of AMD.Targeting HSP90AA1,CHEK1,PSMA4,PSMD4,and/or PSMD8 genes through specific miRNAs or small molecules might potentially alleviate the progression of AMD through attenuating RPE senescence.