The Mechanism Study Of Arabidopsis RH20 Regulating DNA Damage Repair

DNA damage response(DDR)is an important regulatory mechanism for maintaining genomic tability when organisms are exposed to endogenous or exogenous DNA damage factors.Plants are vulnerable to DNA damage due to their fixation and forced dependence on photosynthesis.Therefore,the existence of DNA damage repair mechanism in plants is particularly important.However,the research on the mechanism of DNA damage repair is mainly focused on yeast and animal system,and there is no in-depth research in plants.According to the research status of yeast and animal system,we found that it is a very meaningful research and a good entry point to study the function of homologous genes of animal DDR pathway in plants,which will promote and learn from each other.DDX5(p68)is a key regulator in the animal DDR system,which participates in the DNA damage repair response in animals by regulating the function of p53.However,the function of DDX5 homologous gene in plants has not been studied.We found that RH20 in A.thaliana is a homologous protein of DDX5.Therefore,the function of RH20 in DDR pathway in A.thaliana was studied in this paper.(1)RH20 is expressed in all plant tissues,among which the expression in flowers is the strongest.According to the study of protein localization,it was found that RH20 was expressed in cytoplasm and nucleus.In the nucleus,RH20 is enriched in the nucleolus.(2)Bleomycin Zeocin stress caused DNA damage and DNA double strand breaks in plants.It was found that DNA damage had no significant effect on the accumulation of RH20 transcripts,but its protein accumulation was significantly increased.(3)When wild type and rh20 mutants were treated with bleomycin,it was found that there was no significant change in the number of germinated leaves and root development length of rh20 mutants under no stress.After bleomycin treatment,wild-type plants showed obvious phenotype of DNA damage,including a significant decrease in the number of true leaves and a significant decrease in root length during germination.After bleomycin treatment,the number of true leaves during germination decreased by 1 / 2,and the root length shortened more obviously.After staining the cell death region treated with bleomycin with propidium iodide,it was found that the cell death area of rh20 mutant was significantly larger than that of wild-type plants,indicating that RH20 is involved in plant DNA damage repair.(4)In the process of DNA damage repair,the accumulation of genes in the damage repair pathway,such as BRAC1,RAD51 and WEE1,will significantly increase.In order to explore how RH20 participates in DNA damage repair pathway,we detected the expression level of genes in DNA damage repair pathway by fluorescence quantitative PCR and found that there was no significant change in gene accumulation in DNA damage pathway after RH20 mutation.RH20 may not involve in DNA damage repair pathway by regulating the expression of genes in DNA damage pathway.(5)In order to further explore the mechanism of RH20 involved in DNA damage repair,we used immunoprecipitation-mass spectrometry to identify RH20 interacting proteins.It has been found that RH20 protein can interact with more than 200 proteins.The function of proteins in the library was analyzed and several proteins that may be involved in DNA damage repair were screened,such as CDKA1,MAPK12,EIF4A-2 and CDC48 A.(6)Our study found that there was interaction between CDKA1 and RH20.Therefore,RH20 may be involved in the process of DNA damage repair through the interaction with CDKA1.In animals,CDKA1 affects the repair of DNA damage by regulating the phosphorylation of downstream proteins.Further studies have found that the level of phosphorylation of H1Thr3 decreases significantly after RH20 mutation.These results suggest that RH20 may be involved in DNA damage repair by regulating the phosphorylation level of downstream proteins by CDKA1.To sum up,through a series of studies,we found that the homologous gene of DDX5 in plants,RH20,may regulate the phosphorylation of H1Thr3 through the interaction with CDKA1 protein,and then regulate the repair of plant DNA damage.These results are of certain significance to reveal the mechanism of DNA damage repair in plants.