Molecular Mechanism Of P53S Regulating Helicase To Alleviate DNA Replication Stress

Objective:The smooth progress of DNA replication is the key to maintain normal cell growth.Complex DNA structures,such as G-quadruplex（G4）,affect DNA replication and are potential factors inducing DNA replication stress,DNA damage and cell senescence.Regulating replication stress may delay the senescence process and prevent age-related diseases.Our laboratory has previously found that premature senescent cells derived from Werner syndrome acquired the p53^N236S（p53S）mutation and escaped senescence,suggesting that p53S may be an important regulator to delay the aging process.By studying the regulatory effect of p53S on DNA replication-related proteins and G4structure,this thesis elucidates the molecular mechanism of p53S in alleviating cellular DNA replication stress and delaying the process of aging,and provides new ideas for the prevention and treatment of aging and related diseases.Methods:In this thesis,p53 ^N236S knock-in MEFs(p53S,genotype p53^S/S)were used,and p53 knockout(p53null,genotype p53^-/-)and wild type MEFs(WT,genotype p53^+/+)were used as controls.（1）DNA replication-related pathways and genes regulated by p53S were identified by RNA-seq and gene set enrichment analysis（GSEA）;Western blot was used to verify the expression of DNA helicase and DNA replication fork protection complex protein in three cell lines,and to study the regulation of p53S on the expression of DNA helicase and DNA replication fork protection complex protein.（2）The content of G4 structure in three kinds of cells was studied by immunofluorescence;Double immunofluorescence staining was used to detect the co-localization of G4 structure and DNA helicase protein.Western blot and immunofluorescence staining were used to detect the expression of DNA helicase and replication fork protection related proteins in the cells treated with PDS,and flow cytometry was used to detect the cytologic consequences after PDS intervention.Taken together,we investigated the role of p53S in the regulation of G4 structure and the response to DNA replication stress.（3）To investigate the dynamic regulation of p53S on DNA replication fork binding protein complex and its effect on DNA replication process,the proteins were isolated by iPOND and detected by Western blot.Results:（1）p53S up-regulates the expression of DNA helicases and replication fork protection proteins,thereby activating DNA helicase-replication related pathways.RNA-seq data analysis showed that p53S may up-regulate the expression of DNA helicases and replication fork protection proteins,thereby activating DNA replication related pathways.Western blot results showed that compared with WT and p53null cells,the expression levels of DNA helicases and replication fork protective proteins such as Wrn,Blm,Recql4,Timeless,Ddx11,Gins2,Mcm2,Mcm7,Top2a and Fancd2were up-regulated in p53S cells.（2）p53S up-regulates the expression levels of DNA helicases and replication fork protection proteins,promotes the unwinding of G4 structures,and renders cells resistant to DNA replication damage induced by G4 stabilizers.The results of double immunofluorescence staining showed that compared with WT and p53null cells,the content of G4 structure in p53S cells was less,and the expression levels of DNA helicases Blm,Wrn,Recql4,and Ddx11 were up-regulated,and the helicases co-localized with G4 structure,suggesting that p53S up-regulates DNA helicases to promote G4 unwinding.After replication stress induced by PDS,p53S cells showed lessγ-H2AX damage signal and less G2 phase arrest compared with WT and p53null cells,the apoptotic cells caused by PDS were reduced.These data suggest that p53S cells are resistant to DNA replication damage induced by G4 stabilizers.Interestingly,Western blot results showed that DNA helicase expression was down-regulated in all three cell lines after PDS treatment,but the DNA helicase level in p53S cells was still higher than that in p53null and WT cells.（3）p53S promotes the recruitment of DNA helicases to the replication fork to ensure the DNA replication process.The proteins that bind to the DNA replication fork in three cell lines were analyzed by iPOND technique.The results showed that more DNA helicases such as Timeless,Ddx11,Gins2 and Mcm7 were recruited to the replication fork in p53S cells than in WT and p53null cells,thus promoting the formation of DNA replication fork and DNA replication process.Conclusions:（1）p53S up-regulates the expression of DNA helicases and facilitates the recruitment of these helicases to DNA replication forks,facilitating the unwinding of the G4 structure and ensuring the smooth progress of DNA replication at fragile chromosome sites.Compared with p53 null cells（loss of function）,the regulation of helicase and G4 structure is a new gain of function after p53S mutation.（2）p53S may alleviate the senescence phenotype of premature aging diseases by regulating the replication stress of helicase.This study lays the foundation for screening p53S-like drugs for the treatment of premature aging diseases.（3）Our data also suggest the potential use of G4 stabilizers in the treatment of rapidly growing tumors,especially those with aberrant oncogene activation and increased DNA replication initiation.