The Research On Identification And Screening Of PprM Target DNAs In Deinococcus Radiodurans

Background and Objective: Deinococcus radiodurans is one of the most radiation-resistant organisms known on the earth. It is highly resistant to ionizing radiation, ultraviolet radiation, H2O2, desiccation and other physical and chemical DNA damaging agents. Previously some researches have shown that PprM plays an important role of extreme radiation resistance in Deinococcus radiodurans. Bioinformatics analyses showed that the PprM protein shares high homology with the cold shock protein and contain CSD domains(“Cold-shock” DNA-binding domain),which suggested that PprM might act as a DNA binding protein and combine with the target DNAs to exert relevant biological effects. This study was intended to study of protein-DNA interactions in vivo which applied the currently best method chromatin immunoprecipitation(ChIP)to screen the target DNA interaction with PprM protein, and to explore the regulatory relationship between them.Methods: The HA-pprM gene was amplified by PCR from plasmid DNA of pGEX-6p-1-pprM(about 438 bp) and inserted into shuttle expression vector pRADK to construct pRADK-HA-pprM. Confirmed the correctness of the inserted sequences by agarose gel electrophoresis,restriction enzyme digestion and gene sequencing. The recombinant plasmid was transformed into Deinococcus radiodurans the pprM gene deletion mutant strain and the recombinant protein was analyzed by Western blot. Finally, we used chromatin immunoprecipitation(ChIP) to screen PprM protein's target DNA.Results: Succeeded in constructing the pRADK-HA-pprM shuttle expression vector without any mutation. We discovered that the HA-PprM recombinant protein could be highly expressed in complemented strain and was confirmed by Western blot. We successfully screened three target DNAs by ChIP technology.Conclusions: Successfully constructed shuttle expression vector pRADK-HA-pprM and the pprM complemented strain and screened 3target DNAs which interacted with Ppr M protein by ChIP PCR.Confirmed PprM protein can binding the promoters of pprI ? pprA and recA under 2kGy irradiation.