| Deinococcus radiodurans is one of the most radiation-resistant organisms on the earth, and has drawn a lot of attention in the last several decades, because it shows the extreme resistance to ionizing radiation, UV-ray, desiccation and a variety of DNA damaging agents. D. radiodurans is able to reconstruct the chromosomal fragments from hundreds of double strand DNA breaks (DSBs) during post-irradiation incubation without any mutation. The ability to survive the potentially damaging effects of ionizing and ultraviolet irradiation and desiccation can be the result of three mechanisms: prevention, tolerance, and repair. D. radiodurans efficiently coordinates their repair from ionizing radiation through a complex network of DNA repair and metabolic pathway switching. Therefore, it is essential to demonstrate the function and the biochemical basis of these unknown function novel genes, which are involved in DNA repair and response to environmental stress in D. radiodurans for our understanding of the DNA repair mechanism and extreme radioresistance.RecA is central to homologous recombination repair of irradiation-induced double-strand breaks in D. radiodurans chromosome, and the recA gene mutant is very sensitive to ionizing radiation. D. radiodurans is absent of the classical SOS response pathway mediated by LexA. D. radiodurans RecA is able to mediate the proteolytic cleavage of D. radiodurans two LexA homologs, however, the LexA protein from D. radiodurans is not involved in RecA regulation following exposure to ionizing radiation, indicating a different mechanism which may attribute to extreme radioresistance involved in this pathway.Recently, a radiation sensitive D. radiodurans was isolated from forty natural mutants strain by Hua and his colleagues. Preliminary study reveals that the radiation sensitivity of the mutant resulted from an ISE insertion mutation in the DR0167 gene (named pprI) [Hua et al., 2003]. We will further characterize the novel DNA repair gene pprI by using molecular biological methods: (1) Study on the regulation machenism and some relative biological questions of the pprI gene; (2) Study the relationship between the domains and function of the PprI; (3) Proteomics analysis of the regulation network of the PprI responded to ionizing radiation.1. A pprI gene disruptant (YA1) was constructed generated by transforming a disruption plasmid (containing a chloramphenicol resistance gene induced the promoter of catalase A gene) into KD8301 using the direct insertional mutagenesis technique. The resulting pprI mutant YA1 is very sensitive to γ-ray irradiation: It is predicted that the pprl may be a novel DNA repair gene response to irradiation. To further examine the physiological function of the gene, the three genes pprI, recA and pprA were cloned and the PprI, RecA and PprA were expressed and further purified. Besides, the polyclone antibodies of PprI, RecA and PprA against rabbit were prepared. Immunoblotting assays show that, in response to radiation stress, PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme activities of catalases.2. To evaluate whether PprI also functions in the radioresistance in other organisms, a shuttle vector with D. radiodurans pprI gent was transformed into E. coli. The expression of PprI protein alsosignificantly enhanced the radiation resistance and scavenging ability of free radicals by inducing in the enzymatic activities of catalase E. Although PprI has no significant similarity to any previously characterized proteins in E. coli, the expression of this exogenous gene in E. coli may turn on a unique regulatory pathway and up-regulate DNA protection and damage repair effectors in response to high dose irradiation in a similar manner to that conserved in D. radiodurans.3. From NCBI DNA sequence analysis, pprI is the first gene in a four-gene cluster (DR0167-DR0170) and apparently forms an operon with succedent cistrons (enzyme) folP (EC 2. 5.1.15),fol5 (4. 1. 2. 25), and folK(EC 2. 7. 6. 3) homologs, which are re...
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