Functional Analysis Of Two DNA Damage And Repair-Related FEN/FEN-Like Nucleases

DNA damage and repair is an important self-defence mechanism.It can help rebuild DNA structure when cells suffer from serious injury with repair proteins and other related factors.Nuclease is a kind of repair proteins which can degrade DNA fragments and resect DNA strand end for following repair.FEN1 is an endonuclease protein revolved both in repair and replication process.It takes part in Okazaki maturation and flap DNA degradation in long-patch base excision repair(BER)pathway.Structure and function of FEN1 is conserved across the nature.But in Deinococcus radiodurans(DR),a model organism for DNA damage and repair research,there is no homologue proteins for FEN1.And N-terminal of DNA polymerase I in DR has similar amino acid sequence with FEN1.The same is ture for other bacteria.We analysed phosphorylation,acetylation and succinylation of HeLa cells before and after UV radiation using quantitative proteomics technique.Confirming the conserved amino acids sites in FEN1 acetylated or not can influence FEN1's function and genome stability.We also analysed the functinons of DR DNA polymerase I' N-terminal and confirmed its impotance for cell survival under UV and ? radioation.Results are as follows:1.We obtained 1551 newly analysed phophrylation sites.Phosphorylation sites of DNA damage and repair proteins are found to change evidently.There are overlap proteins and sites between acetylation and succinylation,especially in tricarboxylic acid cycle.UV radiation influence these proteins' modification.2.We analysed sites K252/254 mutation is an important acetylation amino acids for FEN1 nuclease function.Mutated FEN1 protein in K252/254 diminishes nuclease function and increase cell sensitivity to UV radiation.3.Biochemical experiment confirmed that N-terminal of DNA polA in Deinococcus radiodurans species has nuclease and FEN functions.Mutated conserved metal-combined sites D119/120 decreases its nuclease activity.Full-length drPolA protein dosen't show polymerase activity in vitro,but D119/120 A mutated protein and N-truncated drPolA can both perform polymerase activity.DR cells knocked-out the N-terminal of polA gene becomes sensitive to UV and ? radioation.Indicating the N-terminal of drPolA is important for DNA repair in cell survival.