Regulation Of DNA Damage Repair By CRISPR Activating Protein Csa3a In Sulfolobus Islandicus

CRISPR-Cas(Clustered Regularly Interspaced Short Palindromic Repeats and crispr-associated genes)provides an acquired immunity against invasive genetic elements in prokaryotes.The immunologic function of CRISPR-Cas system is strictly regulated by transcription factors and signal molecules.In the Sulfolobus islandicus REY15 A,CRISPR-associated factor Csa3 a triggers CRISPR na?ve adaptation(de novo spacer acquisition),acquisition of new spacers and activates CRISPR RNA transcription for the immunity.On this basis,we focused on the regulation of CRISPR-associated protein Csa3 a on DNA damage response(DDR)system,and the following results were obtained:(1)According to transcriptome and reporter gene data,this study found that deletion of csa3 a gene down-regulated the DNA damage response(DDR)genes,including ups and ced operons,and cdc6-2 and tfb3,which responded to UV-caused DNA damage,in S.islandicus.(2)EMSA and SPR experiments demonstrated that Csa3 a factor bound the promoters of above DDR genes,combining the transcriptome data indicating the transcriptional activation role of Csa3 a for these genes.(3)Optical microscopic analysis showed that compared with wild-type strains,the deletion of csa3 a gene can significantly inhibited the cell aggregation mediated by the Ups system under the treatment of DNA damage agents,and reduce the survival rate of csa3a-deleted strains.Flow cytometry study and survival rate analysis revealed the csa3 a deletion strain was more sensitive to the DNA damage reagent and the cells were more likely to produce DNA fragments..(4)The results of the DNA repair experiment showed that the Ced system can mediated DNA repair of the DNA damage,which caused by CRISPR interference at the targeted cas5 gene locus,and the deletion of csa3 a gene can significantly reduce the repair efficiency.In summary,this study revealed the relationship between the CRISPR Cas system and DNA damage repair mediated by Csa3 a protein,laying a foundation for further research on the relationship between the self immunity of CRISPR Cas system and maintenance of genomic stability.