Function Research On PprI GAF Domain In Deinococcus Radiodurans

Deinococcus radiodurans is a bacterium known for its extremely high tolerance to environmental hazards including ionizing radiation(IR),ultraviolet(UV)radiation,desiccation,oxidative stress and mutagens,and it is a model organism used to study DNA damage repair.Deinococcus radiodurans is famous for its ability to repair massive DNA damage depending on efficient DNA repair pathways.PprI is the switch of DDR(DNA damage response)in Deinococcus radiodurans,and the PprI-DdrO pathway mediates a new DDR pathway different from the classical SOS response in E.coli.As the core of PprI-Ddro pathway,PprI plays an important role in DDR of Deinococcus radiodurans.The crystal structure of IrrE(PprI in Deinococus deserti)has been analysised which contains three domains.The GAF domain located in C-terminal of PprI shares the same structure with other bacteria in Deinococcea.The present study showed that GAF domain acts as signaling molecules receptor,which indicated that GAF domain was essential in PprI-DdrO pathway.The aim of this study was exploring the function of GAF domain from PprI.The major results were as follows:1)Through the sequence alignment of protein,we indicated the conservativeness of GAF domain.We showed the comparability of PprI crystal structure from other bacteria in Deinococcea,which illustrated the research of this domain was universal.2)PprI was a key protein in DNA repair of D.radiodurans,containing N-terminal domain,HTH domain and GAF domain.We generated a deletion mutant strain of GAF domain in D.radiodurans.The survival rate of AC(GAF domain deletion mutant strain)dropped two orders of magnitude compared to wild type under the stress of UV and ??which indicated that GAF domain was related to the UV resistance of D.radiodurans.3)Through hydrolyzing DdrO,PprI can switch on the translation of DNA repair genes.In vitro assays with the purified His-PprI203-321(GAF domain)and His-PprI1-203(PprI without GAF domain)showed that PprI203-321 do not have the ability to hydrolyze DdrO and have merely effect to the protease activity of PprI1-203.4)The interaction between PprI and RNA polymerase ? subunit was explored through yeast two-hybrid system.Deletion of GAF domain caused PprIT176A lost its interaction with RNA polymerase,which indicates that GAF domain was involved in the recruitment of RNA polymerase ? subunit.5)We compared the structure of GAF domain from D.radiodurans and D.deserti.Both of them had a cavity domain,and it's supposed to involve in signal transformation as signal molecule receptor.6)We analyzed the interaction of CAF domain between cGMP/cAMP by ITC(Isothermal Titration Calorimetry)assays.The results showed that GAF protein binds cGMP but not cAMP.This study explored the functions of GAF domain from drPprI both in vivo and in vitro.We determined that GAF domain acts a core role in the UV and ? resistance pathway of D,radiodurans through affecting recruitment of RNA polymerase.We inferred that GAF domain may bind cGMP to response to upstream DNA damage.The research of this domain provide basics for further study of PprI-DdrO response system.