Nuclease EXO1 And CtIp-mediated DNA Damage Repair Regulated By Protein Kinase DNA-PCKs And PLK1 Respectively

There are three important factors,which can affect the selection of DNA double strand breaks repair pathways,includingthe type of cells,the cell cycle and the end resection.After treating withionizing radiation to eukaryotic cells,Non Homologous End Joining(NHEJ)repair pathway master the majority of damage repair,because it is sensitive and quick,meanwhile it does not require specific chromosome morphology,however NHEJ is alsoa error prone repair;As for Homologous Recombination(HR)repair pathway,considering it requires sister chromatid or repetitive sequence as homologous repair templates,HR belongs to accurate repair which mainly happened in S phase and G2 phase.Furthermore,EXO1 and CtIP are the key nuclease of HR pathway,the broken DNA ends firstly identified by the MRN complex,MRE11 and Ct IP endonuclease work together to generate a short 3?-ssDNA tail,then the extensive resection was carried by EXO1/Dna2 exonuclease to generate a 3?-ssDNA overhang(>10 bp),therefore the single strand DNA ends can be bound by RPA instead of DPK complex,as a result,the progress of end resection stimulates HR repair pathway.Objective:(1)To figure outthe mechanism of DNA-PKcsregulates the stability of EXO1 to mediate the selection between the two differentDNA double strand breaks repair pathways-HR and NHEJ.(2)To predicta interacting protein of CtIP in PrePPI database and further study the mechanism between them.Methods:(1)After adding specific inhibitor of DNA-PKcs,or siRNA to knockdown DNA-PKcs expression in Hela cells,we detectedthe expression of EXO1 protein,mRNA and ubiquitination by western blot,RT-PCR,flow cytometry,CO-immunoprecipitation and so on,to study the molecular regulatory mechanism.(2)To predict CtIP protein interactions and further study the interaction methods to find CtIP interacting proteins in PrePPI database,pick a protein of interest and by co-immunoprecipitation methods to verify the interaction between construct truncated body Looking interaction site,Frodock 2.0 tools docking interaction between the two,Finally,immunofluorescence assay was observed between the two co-localization.Results:(1)Pretreating with specific inhibitor ofDNA-PKcs and siRNA in HeLa cells,We found DNA-PKcs can mediates HR pathway in a certain way.After pretreated with NU7026 and NU7441 for 6 and 8 hours to HeLa cells,Western blot detection of a higher level of EXO1 protein;Meanwhile,we detected inhibition of DNA-PKcs by Nu7441 or NU7026 also delayed EXO1 degradation and ubiquitination;Moreover,Previous studies have revealed that NU7441 and NU7026 inhibited the phosphorylation of s2056 site in DNA-PKcs,compared with a higher level of DNA-PKcs(phospho S2056),the EXO1 protein has lowest level in G0/G1 phase.(2)Results PrePPI database to find a PLK1 is predicted scores of ranking Ct IP interacting proteins,further illustrated by the overexpression of two endogenous proteins and co-immunoprecipitation assay two protein interactions.By building PLK1 and CtIP different truncated body further validate the interaction between the two.The KD domain and middle sequence of PLK1 has a interaction with CtIP,at the same time,the17-160 amino-acid residues in N terminus of CtIP can interact with PLK1.Moreover,Frodock 2.0 tool displays CtIP docking can be formed with a total length of PLK1,N and C-terminal truncation different body.Conclusion:(1)In summary,we found here that DNA-PKcs is an upstream regulator of EXO1 ubiquitinaion and degradation,indicating that it's kinase activity is required for the regulation of EXO1 stability;Meanwhile,the knockdown of DNA-PKcs can also affect the the level of expression of EXO1,suggesting that DNA-PKcs can regulates EXO1 by own kinase activity and expression to mediated the selection between HR and NHEJ repair pathway in cells.(2)Conclusions from bioinformatics,molecular biology,cell biology and structural biology point of view,we can prove that PLK1-CtIP interact,both might beinvolved in regulating DNA damage repair and cell cycle regulation.