The Acetylation Modification Of DNTP Hydrolase SAMHD1 Involved In DNA Damage Repair Response

Objective: In the process of life,cell homeostasis will always face the threat from harmful factors,genetic factors and pathological factors inside and outside the body,which will lead to DNA damage and "imbalance" of genome integrity and stability.In the process of development,living individuals have acquired a series of cell homeostasis maintenance mechanisms,including DNA damage repair response(DDR),cell cycle checkpoint,apoptosis and necrosis,cell aging and autophagy,which are responsible for maintaining cell homeostasis.The "dynamic balance" of DNA damage repair response system is an important guarantee to maintain the integrity and stability of genome,and its "imbalance" will lead to the occurrence of disease.In recent years,it has been found that post-translational modification of proteins can regulate DNA damage repair response and affect genome integrity and stability.The post-translational modification of proteins mainly includes methylation,glycosylation,acetylation and phosphorylation.To explore the regulation of post-translational modification of proteins on DNA damage repair is a hot topic in this field.SAMHD1 was originally found in the cDNA library of human dendritic cells,and its protein structure includes SAM domain and HD domain.In recent years,the research focus of SAMHD1 has been mainly on its antiviral function.SAMHD1 is the first deoxyribonucleoside triphosphate hydrolase found in eukaryotic cells.It can hydrolyze d NTP into nucleoside and triphosphate,which play a key role in regulating the dynamic balance of dNTP content in cells.The latter is the necessary raw material molecule for DNA damage repair.SAMHD1 can be phosphorylated at Thr592,which affects the content of dNTP and DNA damage repair.It was found that the mutation of SAMHD1 was closely related to Aicardi-Goutières syndrome(AGS).The increase of dNTP level and chronic DNA damage were detected in fibroblasts of patients.In chronic lymphocytic leukemia(CLL)patients,SAMHD1 was found to affect cell proliferation and survival during DNA damage induction.It has been reported that the acetylation modification of SAMHD1 may be involved in the tumorigenesis,suggesting that SAMHD1 is involved in DDR,and its acetylation modification may play a regulatory role in DDR.These findings suggest a new link between SAMHD1 and DNA damage in the pathogenesis of the disease.The team confirmed in a series of previous research results that DNA damage induced the level of phosphorylation of SAMHD1.The purpose of this study is to explore the new epigenetic modification acetylation regulation of SAMHD1 protein and its effect on DNA damage repair response,so as to provide a new perspective for elucidating the molecular mechanism of DNA damage repair and the mechanism of genome homeostasis maintenance,and provide a new strategy for the treatment of clinical DNA damage related diseases.Methods: 1.In this study,we first used pan acetylated antibody to complete the process of immunocoprecipitation in human embryonic kidney 293(HEK293)cell,and confirmed that SAMHD1 protein can be acetylated by western blotting,and found its acetyltransferase.2.Cells were stimulated with DOX to simulate DNA damage.Pan acetylated antibody was used to complete the process of immunocoprecipitation,then the acetylation modification level of SAMHD1 was detected by western blotting.3.Histone deacetylase(HDAC)inhibitors Trichostatin A(TSA)and Nicotinide(NAM)were used to treat HEK293 cell,then the acetylation and phosphorylation(T592)modification level of SAMHD1 were detected by western blot.4.According to the literature reports,the K405 site is the acetylation modification site of SAMHD1 protein.Therefore,we constructed pcDNA4/flag-myc-his-SAMHD1(K405R)plasmid and stable transfer HEK293(Cas9-Samhd1)cell respectively by the construction of point mutation plasmid technology and the clustered regular inter spaced short palindromic repeats associated protein 9(CRISPR-Cas9)technology.PcDNA4/flag-myc-his-SAMHD1(WT)and pcDNA4/flag-myc-his-SAMHD1(K405R)plasmids were transfected into HEK293(Cas9-Samhd1)cell,then the acetylation modification level of SAMHD1 was detected by western blotting.DNA damage DOX(0.5?M)was stimulated,then the phosphorylation modification level of SAMHD1 and the level of DNA damage repair related marker protein ?H2AX(Ser139/Tyr142)were detected by immunofluorescence and western blotting.5.Binding between SIRT2 and SAMHD1 was detected for the first time by immunocoprecipitation and protein binding in vitro.After stimulation of DNA damage DOX(0.5?M),the binding of SIRT2 and SAMHD1 were detected by immunocoprecipitation in HEK293 cell.6.HEK293 cell was given SIRT2 protein specific inhibitor AGK2,then the acetylation and phosphorylation modification level of SAMHD1 were detected by western blot.7.DNA damage DOX(0.5?M)was stimulated in stably transformed HEK293(shSIRT2)cell,then the phosphorylation modification level of SAMHD1 and the level of DNA damage repair related marker protein ?H2AX(Ser139/Tyr142)were detected by western blot.8.SIRT2 overexpression plasmid was transfected into HEK293 cell and DNA damage DOX(0.5?M)was stimulated,then the phosphorylation modification level of SAMHD1 and the level of DNA damage repair related marker protein ?H2AX(Ser139/Tyr142)were detected by western blot.Results: 1.SAMHD1 protein can be acetylated and its acetyltransferase was GCN5.2.When DNA damage DOX(0.5?M)was stimulated,the acetylation modification level of SAMHD1 decreased.3.After the treatment of TSA and NAM,the acetylation modification level of SAMHD1 increased significantly,and the phosphorylation modification level of SAMHD1 decreased.4.The acetylation modification level of SAMHD1 of HEK293(Cas9-Samhd1)cell transfected with K405 R plasmid was lower than that of HEK293(Cas9-Samhd1)cell transfected with WT plasmid.When DNA damage DOX(0.5?M)was stimulated,the phosphorylation modification level of SAMHD1 in HEK293(Cas9-Samhd1)cell transfected with K405 R plasmid was lower than that in HEK293(Cas9-Samhd1)cell transfected with WT plasmid,but the level of DNA damage repair related marker protein ?H2AX(Ser139/Tyr142)was higher than that in HEK293(Cas9-Samhd1)cell transfected with WT plasmid,suggesting that DNA damage was aggravated 5.SAMHD1 and SIRT2 can combine with each other in vivo and in vitro,and the combination of them was enhanced under the stimulation of DNA damage DOX(0.5?M).6.After treatment of SIRT2 protein specific inhibitor AGK2,the acetylation modification level of SAMHD1 protein increased,while the phosphorylation modification level decreased.7.In stably transformed HEK293(shSIRT2)cell,DNA damage DOX(0.5?M)was stimulated,then the phosphorylation modification level of SAMHD1 decreased,and the level of DNA damage repair related marker protein ?H2AX(Ser139/Tyr142)increased,suggesting that DNA damage was aggravated.8.SIRT2 was overexpressed in HEK293 cell and DNA damage DOX(0.5?M)was stimulated,then the phosphorylation modification level of SAMHD1 was increased,and the level of DNA damage repair related marker protein ?H2AX(Ser139/Tyr142)was decreased,suggesting that DNA damage was reduced.Conclusion: 1.SAMHD1 protein can be acetylated.2.Under the pressure of DNA damage,the acetylation modification of SAMHD1 protein can regulate its phosphorylation modification,and then affect the DNA damage repair response.3.SIRT2,a deacetylase,may be involved in the regulation of DNA damage repair by regulating the acetylation modification of SAMHD1 protein.