Study On PprI Protease-mediated DNA Damage Response Mechanism In Deinococcus Radiodurans

The extremophilic bacterium Deinococcus radiodurans exhibits a miraculous resistance to extreme ionizing radiation、ultraviolet light、chemical mutagens. Deinococcus radiodurans is one of the most anti-radiation organism discovered by far due to its efficient DNA repair mechanism, so it is used as an ideal model system for studying DNA repair. Although in recent decades scientists have tried to crack this mystery and made a lot of breakthroughs, its underlying repair mechanism is still poorly understood.In previous studies, PprI, an essential protein for the extreme resistance, was characterized, which acts as a general switch orchestrating the expression of a number of proteins involved in pathways for cellular radio-resistance. The PprI structure shows that this protein is consisted of three domains, including a zinc peptidase like domain, a helix-turn-helix (HTH) motif and a GAF-like domain. Recently, we has been preliminarily studied the HTH motif and confirmed that its DNA binding is also required for DNA repair in Deinococcus radiodurans. However, the functions of the other motifs are still unclear. Based on this background, in this study, we investigated the zinc-finger protease activity of PprI, and discussed the roles of the protease and its substrate playing in DNA damage response pathways. The detailed results were as follow:1. We discovered the protease substrate of PprI, DdrO. In vitro, high pure PprI and DdrO purified through AKTA Purifier were mixed, detected by SDS-PAGE, indicating that DdrO can be cleaved by PprI. This result was further demonstrated by measurement of molecular weight for the digested products and carboxyl terminal sequencing by MS. In vivo, We detected the DdrO bands of wildtype strain R1and pprl deletion mutant after irradiation, found that the bands of wildtype strain R1disappeared in one hour after irradiation, while the band in YR1still existed. So it proved that the change of DdrO after irradiation depends on PprI.2. The PprI cleavage of DdrO is dependent on Mn(2+) ion. Residual divalent metal cation was first removed from the puried using EDTA. Then various metal ions were added before the reaction was initiated by adding the purified DdrO, which showed that the only metal ion for activating this proteolytic reaction is Mn(2+). Further, mutations of four residues (HEXXE) mediating the metal binding (His-118, Asp-119, His-122, Asp-149) abolished the cleavage activity, although Mn(2+) ion was added.3. We identified proteolytic cleavage site and sequence of PprI. According to the molecular mass detection and C-terminal sequencing of enzymatic product, it was speculated that the proteolytic cleavage site is between Leu-108and Arg-109. A series of mutants of DdrO around the cleavage site were constructed, tested the sensitivity to the PprI protease, following to set up a supposed model of the cleavage.4. DdrO protein can specifically bind to the promoters of DDR genes in vivo or in vitro. We proved that DdrO can bind to the promoter region of DDR genes using EMSA, including ddrO itself in vitro. We further applied ChIP assay to confirm its binding ability in vivo and found that rabbit anti-DdrO antibody could enrich more than four times of those promoters than nonspecific antibody, indicating that the promoters of DDR genes are bounded by the DdrO in vivo.5. RDRM sites in the promoters are necessary for DdrO binding. The promoter regions without the17-bp RDRM sequences amplied by PCR were tested using EMSA. The split promoter regions lost the capacity of binding to DdrO. We further examined the minimum binding sequence of DdrO in PrecA, and confirmed that RDRM site is the direct binding site of DdrO.6. The cleavage of DdrO by PprI is enhanced by the substrtate DNA binding. Lots of PrecA and PddrO was puried, bounded enough with DdrO, then added the same quantity of PprI. The result of cleavage reactions showed that the binding to specific double DNA can greatly enhanced the cleavage efficiency. What’more, EMSA was applied to test the binding between DNA and DdrO in the presence of PprI. The band shift disappeared, indicating that the binding was abolished.7. We analyzed the phenotype of a ddrO-site disruptant. A ddrO-site disruptan designated R109E was constructed, which was very susceptible to y radiation、UV、 and H2O2. In vivo, DdrO bands in strain R109E were still present and uncleavable after radiation, which is corresponding to the character of YR1.8. RNA transcription inhibition of DDR genes is removed by PprI after radiation. Using qRT-PCR, RNA transcription levels of DDR genes in R1、YR1、R109E and CR109E were studied at the different recovery time points after irradiation. On the basis of intuitionistic analysis of the transcription and expression of ddrO gene and pprl gene, We revealed that under the normal growth environment, RNA transcription of DDR genes was repressed by DdrO, but the inhibition would be immediately abolished after exposing to irradiation.To sum up, PprI protein and its substrate, DdrO protein, are essential to DNA damage response. After irradiation, the subtle interaction between them mediates a novel super efficient DNA damage response and repair pathway, differing from the classic LexA-RecA-mediated SOS response system found in radiation-sensitive bacterium E. coli. The Pprl-mediated pathway in the D. radiodurans greatly contributes to its extreme radio-resistance and is anticipated to add knowledge to DNA damage response models.