Tudor-SN Protein Function In The DNA Damage Response

Objectives:Genome integrity is continuously challenged by DNA lesions,many thousands of which arise in each human cell every day.While the majority of these lesions occur as byproducts of normal cell metabolism or DNA replication,they are also induced by radiation and toxic environmental chemicals.To deal with the fundamental problem of genomic erosion,a sophisticated network of DNA damage-response(DDR)systems has evolved.These include activation of a cell cycle checkpoint,execution of DNA repair,or when the damage is severe,initiation of apoptosis.Tudor staphylococcal nuclease(Tudor-SN)is a highly conserved and ubiquitously expressed multifunctional protein.It has been identified as co-activators of a variety of transcription factors such as STAT6 in the nucleus,also participate in the pre-mRNA splicing and processing,stress granules'formation.It is important to note that CoaSt6 and PARP-14,subsets of Poly(ADP-ribose)polymerases(PARP),can append ADP-ribose to Tudor-SN.The data of mass spectrometry indicated that Tudor-SN could bind with PARP-1.Our main purpose of this study is to explore the potential role of Tudor-SN in DNA damage response and its possible function mechanism during the process.Methods:Part ?:The interaction of Tudor-SN and DNA repair proteins.This part was divided into two aspects:on one hand,using mass spectrometry to found the Tudor-SN protein can bond with PARP-1;On the other hand,using immunoprecipitation and GST-pulldown assay to detected the interactions of Tudor-SN and PARP-1 in vivo and in vitro.Part ?:Using immunoprecipitation to detect Post-translational modification of Tudor-SN protein in DNA damage response.Part ?;The affections of Tudor-SN on apoptosis induced by DNA damage.First,using flow cytometry analysis to detect if Tudor-SN protein affect apoptosis induced by DNA damage inducer.Second,Western Blot and immunoprecipitation assays were performed to detect apoptosis markers in WT or KO cells.Third,observing the affections of Tudor-SN on cell proliferation by cloning assay.Part ?:The affections of Tudor-SN on histone modification in DNA damage response.On one hand,to observe the effect of Tudor-SN protein on H2AX phosphorylation using Western Blot method,H2AX activation double staining detection and immunofluorescence;on the other hand,to learn how Tudor-SN influence histone acetylation by Western Blot,immunoprecipitation assay and chromosome relaxation assay.Part V:The role of Tudor-SN in ATM-dependent DNA damage signaling pathway.Western Blot and immunoprecipitation assay were performed to detect the regulation of Tudor-SN on ATM-dependent DNA damage signaling pathway.Results:1.The mass spectrum result showed Tudor-SN protein can bond with PARP-1 protein;Tudor-SN can combine with DNA damage repair proteins such as PARP-1,XRCC1,DNA Polymerase beta in vivo;Tudor-SN interacted with PARP-1 protein in vitro.2.Tudor-SN became modified by phosphorylation,Poly(ADP-ribosyl)ation and ubiquitination in DNA damage response.3.The affections of Tudor-SN on apoptosis induced by DNA damage.Tudor-SN knockout induced apoptosis and inhibited cell proliferation or cell viability obviously.4.Tudor-SN enhanced H2AX phosphorylation and promoted H3K9ac expression in DNA damage response.Upon DNA damage,Tudor-SN recruited more histone acetyltransferase Gcn5;Tudor-SN knockout blocked chromatin accessibility to MNase.5.Tudor-SN can promote the phosphorylation of ATM protein kinase and activate ATM mediated phosphorylation pathway;Tudor-SN mediated the combination of ATM and NBS1.Conclusion:1.Tudor-SN combines with PARP-1,XRCC1,DNA Polymerase beta and other DNA repair proteins,forming a complex to regulate DNA damage response.And DNA damage induces Post-translational modifications of Tudor-SN,such as phosphorylation,Poly(ADP-ribosyl)ation and ubiquitination.2.Tudor-SN has a negative influence on apoptosis upon DNA damage.3.Tudor-SN regulates histone modification in DNA damage response.Tudor-SN enhances H2AX phosphorylation;Tudor-SN promotes H3K9ac expression by recruitment histone acetyltransferase Gcn5;Tudor-SN is required for chromatin relaxation in MEFs upon DNA damage.4.Tudor-SN activates ATM-dependent DNA damage signaling pathway.