The Role Of Human DNA Polymerase Polδ And DDR-Related Proteins In UV-C-Induced Cell Damage And Apoptosis

Human DNA polymeraseδ(polymeraseδ,Polδ)is a heterotetramer complex,which is composed of four subunits p125,p50,p68,and p12.Polδis one of the main polymerases in the DNA replication process.It has 5’-3’polymerase catalytic activity and 3’-5’exonuclease activity.Polδplays a momentous role in DNA replication,damage repair,cell cycle maintenance,chromosome structural integrity and genetic stability.Human genomic DNA can be damaged by a variety of exogenous and endogenous factors,such as ionizing radiation,ultraviolet(UV)radiation,reactive oxygen species,lipid peroxidation products,etc.To deal with DNA damage and keep the genome integrity of human cells,there are a set of defense mechanisms which call DNA damage response(DDR).DDR is a complex multi-directional signal pathway.The damage sensing proteins(ATM/ATR)can sense damage and transmit signals to downstream target proteins(Chk1/p53),thereby activating DNA damage repair pathways and regulating various cellular processes,such as DNA replication,transcription,cell cycle arrest,apoptosis and necrosis.Reported strudies of our laboratory showed that the p12 subunit of Polδof Melanoma cells was degraded by treatment of DNA damage agents HU(hydroxyurea),MMS(methyl methanesulfonate),and APH(Aphidicolin),but the exprssion of the other three subunits almost unchanged.In the apoptotic process induced by calcium ions,the p12subunit is degraded in the early stage,restored thereafter,and then depleted again in the later stage.Polδcould play an important role in DDR and apoptosis.So how do cells respond to UV-C-induced DNA damage?What Polδ-related proteins are involved?Corresponding to degradation of the p12 sunuint,how are the response proteins coordinated regulation?There is still little research and further exploration is needed.In this study,we firstly used CCK-8 assay,cell coutting assay,cell scratch assay,flow cytometry to respectively observe the survival rate,cell migration,cell cycle,and apoptosis of Hela cells after UV-C radiation.The results showed that:1.By CCK-8 and cell counting experiments,we found that Hela cells treated with 20 J/m~2or 30 J/m~2doses of UV-C resulted in a down-regulation of cell proliferation capacity and a decrease in cell number.2.Cell scratch experiments showed that UV-C treatment at dose of 20 J/m~2inhibited the migration of Hela cells.3.The cell cycle analysis revealed that the DNA damage caused by UV-C treatment at dose of 20 J/m~2resulted in the cell cycle arrest at the G1/S phase.4.Apoptosis have been found in Hela cells both in the morphology and flow diagram.However,with the same radiation dose,the apoptosis rate increases with the increase of radiation dose.The preliminary experimental results showed that:UV-C radiation could inhibit the Hela cells proliferation,reduce migration rate and induce cell cycle arrest and apoptosis.5.After Hela cells were treated with UV-C,the DNA polymerase Polδresponded quickly,the degradation of p12 subunit of Polδresulted in the conversion of the Polδ4 to the Polδ3.Then,based on the early findings of this study,we used 20 J/m~2or 30 J/m~2doses of UV-C radiation to induce cell cycle arrest or apoptosis in Hela cells,respectively,and analyzed the expression level of four subunits of Polδand related proteins by Western blot assay.The results showed that:1.In the process of cell cycle arrest induced by 20 J/m~2dose of UV-C,the p12 subunit of Polδwas degraded while the protein levels of other three subunit protein levels almost unchanged,and the phosphorylation level of Chk1(Ser345),a single-stranded DNA damage response protein and its downstream signal molecule,p53(Ser15),increased.2.In the process of UV-C-induced apoptosis at 30 J/m~2doses,the p12 subunit was down-regulated,restored thereafter,and then down-regulated again.Meanwhile,the phosphorylation level of Chk1(Ser345)and p53(Ser15)increased,the expression level of Bax,an apoptotic protein,is up-regulated.The shear activation of Caspase-9 and Caspase-3,the downstream apoptosis-related proteins,increased significantly.3.When the down-regulation of the p12 subunit was inbibited by the proteasome inhibitor MG132at the early apoptosis stage of Hela cells induced by 30 J/m~2dose of UV-C,the hosphorylation level of p53(Ser15)increased,the expression of CD95,PCNA(Proliferation Cell Nuclear Antigen)and Bax increased while the level Bcl-2decreased comparing to the UV-C treatment group.Combined,we thought that UV-C radiation-induced the dynamic,synergistic activation of Chk1,p53,Bax,Caspase-9and Caspase-3,activated the ATR-Chk1-p53 signaling pathway,and resulted in cell cycle arrest and apoptosis.Meanwhile,the degradation of p12 subunit of Polδresulted in the conversion of the Polδ4 to the Polδ3 which is a novel cellular response to DNA damage.This study could contribute to the understanding of the new response mechanism about DNA damage response.It also has valuable implications regarding its potential contribuions to the prevention and therapy of cancer and other diseases.