Important Acylation Modification Sites Of FEN1 Involved DNA Damage Response And Repair

The flap endonuclease 1(FEN1)is a member of Rad2 nuclease superfamily,which is conserved from archaea to humans.FEN 1 can extensively involve in DNA metabolic pathways.The important role of FEN1 in DNA replication,recombination and repair determines its critical role in maintaining genomic stability.Abnormal expression or function of FEN1 can lead to disorders in many life processes in organisms,which will cause serious damage to the organisms.Therefore,the expression of FEN1 in vivo must be regulated strictly,accurately and promptly.Previous studies have shown that post-translational modification plays an important role in regulating the activity,cellular localization and functional stability of FEN 1 protein.The acylation(acetylation and succinylation)modification of FEN1 may play a role in cell metabolism and DNA damage repair,but its specific biological effects and mechanisms are still unclear.In view of this,the function and mechanism of acylation of FEN 1 in maintaining cell genome stability were studied in this paper.Two acylation sites(Lys200 and Lys201)of FEN 1 were identified and validated by constructing mutation cell lines.The enzyme kinetic activity experiments in vitro showed that the EXO and GEN activities of mutation proteins(K200E and K201E)were down-regulated compared with FEN1-WT protein,which decreased by about 40%and 67%,respectively.It suggests the acylation may play a role in DNA damage repair.Cell viability test showed that K200E and K201E mutants were more sensitive to damage factors(UV,MMC,CPT).Further interaction protein detection assay revealed that compared with FEN1-WT protein,acylation defective proteins(K200E and K201E)have significant differences in binding ability with DNA repair-related proteins and cell cycle-related proteins.Immunofluorescence assay showed that the foci of y-H2AX and 53BP1 increased in two acylation deficient cell lines of FEN1.The cell cycle assay showed that under the pressure of DNA damage,the cell cycle of K200E and K201E cells were arrested in G1 phase.In conclusion,our study demonstrated that two key sites of FEN1 regulate their enzymatic activity through acylation modification,affect the DNA damage repair efficiency,and then affect the biological phenomena and possible mechanisms of cell cycle progression and damage sensitivity.And the importance of acylation modification of FEN 1 in maintaining cell genome stability was clarified.