Involvement Of Srx1 In DNA Double-strand Break Damage Response In Saccharomyces Cerevisiae

Genomic DNA in living organisms can be damaged by factors from endogenous or exogenous sources in the cell,leading to genomic instability and inducing the development of cancer and many diseases.Srx1 is an endogenous antioxidant protein in Saccharomyces cerevisiae that resists oxidative stress induced by reactive oxygen species,and its antioxidant function plays an important role in the development of diseases.There are many types of DNA damage caused by reactive oxygen species,including base modifications,basic sites,DNA protein cross-links and DNA strand breaks.Eukaryotic cells respond to these damages by DNA damage repair pathways and DNA checkpoint mechanisms,where the damage repair pathways include base excision repair,nucleotide excision repair,homologous recombination and nonhomologous end joining.It is still unclear whether Srx1 plays an important role in the process of DNA damage and repair.In this study,we used Saccharomyces cerevisiae as a model organism to investigate the regulatory role of Srx1 on DNA damage repair and damage response in Saccharomyces cerevisiae and its molecular mechanism.First,mutants of Srx1 and other eight DNA damage repair-related genes were constructed by gene knockout in wild-type cell W303(take Srx1 as an example,Srx1 stands for Srx1protein;Srx1 stands for Srx1 gene;srx1Δ represents that the Srx1 gene has been knocked out).Then the sensitivity of srx1Δ to different DNA damaging agents was tested by drug sensitivity assay,the results showed that srx1Δ was sensitive to the DNA double-strand break damaging agent zeocin after long-term exposure to DNA damaging agents,it was not sensitive to replication stress caused by hydroxyurea,alkylation damage caused by methyl mesylate,single-strand breaks caused by camptothecin,and oxidative stress damage caused by 4-nitroquinoline-1-oxide;the cell mortality rate under transient zeocin treatment was further detected by methylene violet staining,and the results showed that the cell survival rate of srx1Δdecreased compared with that of wild-type W303,indicating that Srx1 played an important role under either long-term or transient DNA double-strand break damage.The DNA damage rate was detected by real-time fluorescence quantitative PCR,and the results showed that the DNA double-strand break damage rate of srx1Δ strain increased under zeocin treatment,indicating that Srx1 was involved in the damage repair process of DNA double-strand break.The repair of DNA double-strand break damage is selected primarily by whether excision occurs at the break end,and if excision occurs at the end,the damage is repaired by the homologous recombination pathway.The initiation of excision is triggered by the cooperation of the Mre11-Rad50-Xrs2(MRX)complex and the nuclease Sae2,followed by remote excision by the nucleases Exo1 and Sgs1.To investigate whether Srx1 is involved in end resection,the sensitivity of srx1Δsgs1Δ,srx1Δsae2Δ,and srx1Δexo1Δ to zeocin was examined by drug sensitivity assay,and the results showed that all were the same as the sensitivity of srx1Δ to zeocin,and the single-knockout pathway gene effect was similar to double-knockout when both genes were located in the same pathway,suggesting that Srx1 and Exo1,Sgs1,and Sae2 act in the same pathway,indicating that Srx1 is involved in the end resection process and presumably acts upstream of Sae2.The protein interactions were further examined by yeast two-hybridization,and the results showed that there was an interaction between Srx1 and Mre11 and Sae2 thus participating in end resection.As the strand exchange protein Rad51 binds to the single-stranded DNA produced by excision after break end resection for homologous search and strand invasion to repair the damage,this study examined the formation of Rad51 focal points by laser confocal microscopy and found that the end resection defect of srx1Δ inhibited the aggregation of Rad51 on single-stranded DNA during homologous recombination,suggesting that Srx1 is involved in end resection thereby promoting homologous recombination to repair double-stranded break damage.Double-strand break damage activates the DNA damage checkpoint pathway,which is signaled by the sensor kinase Mec1 that senses the damage signal and transmits the signal via the mediator protein Rad9 to the effector kinase Rad53,which is phosphorylated to activate the checkpoint.In this study,we investigated the function of Srx1 in the DNA damage checkpoint.We detected the phosphorylation of Rad53 during damage and repair by Western Blot and found that Rad53 phosphorylation was consistently higher in srx1Δ than in wild type,indicating that excessive DNA double-strand break damage in srx1Δ led to Rad53 hyperphosphorylation and that Rad9Δ had reduced Rad53 phosphorylation in W303 and srx1Δcells,suggesting that Srx1 negatively regulates the Rad9-dependent DNA damage checkpoint.After DNA damage repair is completed,the checkpoint is inactivated by Rad53 dephosphorylation,and the phosphatases Ptc2 and Pph3 regulate Rad53 dephosphorylation through different pathways.This study further investigated whether Srx1 has a function in regulating Rad53 dephosphorylation,Rad53 phosphorylation of srx1Δptc2Δ was found to be consistent with that of srx1Δ by Western Blot experiments,and Rad53 phosphorylation of srx1Δpph3Δ was higher than that of srx1Δ,indicating that Srx1 and Ptc2 act in the same pathway,in parallel with Pph3,to regulate Rad53 dephosphorylation.In summary,the results of this study indicate that the Srx1 gene deletion in Saccharomyces cerevisiae is sensitive to the DNA double-strand break damage agent zeocin and that Srx1 is involved in the DNA double-strand break damage repair process;Srx1 is involved in DNA double-strand break end excision and thus promotes the homologous recombination pathway to repair the damage by interacting with Mre11 and Sae2;the DNA double-strand break damage triggered by the Srx1 mutant results in the negative regulation of Srx1 dependent on the Rad9 DNA damage checkpoint;Srx1 and phosphatase Ptc2 act in the same pathway,in parallel with phosphatase Pph3,to regulate Rad53 dephosphorylation and inactivate the DNA damage checkpoint.