| The extremely radio-resistant bacterium, Deinococcus radiodurans, is characterized by its unusual capability to tolerate numerous DNA damaging agents, including ionizing radiation, ultraviolet, hydrogen peroxide, desiccation and other physical and chemical DNA-damaging agents. However, little is known about the molecular mechanisms responsible for the extraordinary resistance in this organism. In this study, the functions and regulation of these proteins involved in DNA damage repair were investigated. The results as below:1 RadA takes part in the DNA damage repair process in D. radioduransRadA is highly conserved in bacteria and belongs to the RecA/RadA/Rad51 protein superfamily found in bacteria, archaea and eukaryota. In Archaea, it plays a critical role in homologous recombination process due to its RecA-like function. In Escherichia coli, it takes part in conjugational recombination and DNA repair but is not as important as that in archaea. Using PSI-BLAST searches, we found that D. radiodurans RadA has a higher similarity to that in bacteria than that in archaea and eukaryota. Disruption of radA gene in D. radiodurans resulted in a modestly decreased resistance to gamma radiation and ultraviolet, but had no effect on the resistance to hydrogen peroxide. Complementation of the radA disruptant by both E. coli radA and D. radiodurans radA could fully restore its resistance to gamma radiation and ultraviolet irradiation. Further domain function analyses of D. radiodurans RadA showed that the absence of the zinc finger domain resulted in a slightly more sensitive phenotype to gamma and UV radiation than that of the radA mutant, while the absence of the Lon protease domain exhibited a slightly increased resistance to gamma and UV radiation. These data suggest that D. radiodurans RadA does play an important role in the DNA damage repair processes and its three different domains have different functions.2 D. radiodurans RecD has a new role in antioxidant processRecBCD holoenyme plays a critical role in double-stranded DNA breaks in many organisms. In D. radiodurans, this complex is not intact because of the absence of RecB and RecC, but a RecD-like protein does indeed exist. Biochemical analysis has been shown that it is a 5'-3' helicase and has a low processivity. In this work, we constructed D. radiodurans recD disruptants and investigated their possible biological functions. The results showed that disruption of the recD gene of D. radiodurans resulted in remarkably increased sensitivity to hydrogen peroxide and slightly decreased resistance to ultraviolet radiation, but had no apparent effect on the resistance to gamma radiation compared to the wild type. Furthermore, complementation experiments showed that E. coli RecD, helicase domain or N-terminal domain of D. radiodurans RecD couldn't individually restore the resistant phenotype to hydrogen peroxide of the D. radiodurans recD disruptant, whereas the complete D. radiodurans RecD protein and co-expression of the helicase domain and the N-terminal domain of D. radiodurans RecD could, indicating that the integrity of D. radiodurans RecD is required for this function. Further studies showed that D. radiodurans RecD took part in antioxidant process by stimulating catalase activity and reactive oxygen species scavenging activity in D. radiodurans. These results suggest that D. radiodurans RecD has a new role in the antioxidant pathway.3 D. radiodurans doesn't have a classical SOS responseLexA and RecA play a crucial role in SOS response in E. coli. In classical SOS response, LexA functions as a repressor to regulate the expression of many genes including RecA after DNA damage occurs. Previous studies showed that both LexAl and LexA2 are not involved in RecA induction in D. radiodurans. In this study, we constructed a double mutant of lexAl and lexA2. Results showed that mutation of lexAl and lexA2 resulted in an apparent decrease in growth rate and an increased resistance to ultraviolet and hydrogen peroxide compared with wild type Rl and two single mutants. However, this double mutant did not exhibit any remarkably increased constitutive expression of RecA under normal conditions, just like wild type Rl and two single mutants, suggesting that neither LexAl nor LexA2 is involved in the induced expression of RecA. Further transcriptional assay showed that LexAl and LexA2 together participated in many regulatory pathways involved in cell division, protein synthesis, antioxidant process, transcription regulation andother mechanisms. These results indicate that there should be a new mechanism bywhich these damage-induced proteins are regulated in D. radiodurans.4 PprI regulates the induced expression of RecA and PprA via antagonizingRecXRecA plays a central role in homologous recombination repair to mend double-strand DNA breaks when DNA is highly damaged and PprA is involved in the non-homologous end-joining repair pathway. To date, little is known about the biochemical mechanism controlling the expression of D. radiodurans RecA and PprA. Our previous work showed that PprI is a positive factor and RecX is a negative factor in the transcriptional regulation of D. radiodurans RecA. Here, we found that the double mutation of D. radiodurans pprI and recX could restore the resistance of the pprl mutant to ultraviolet and hydrogen peroxide but not to gamma radiation. Furthermore, complementation of the pprI mutant by D. radiodurans RecA also could fully recover the resistance of the pprI mutant to ultraviolet and hydrogen peroxide, however, D. radiodurans PprA couldn't compensate all three kinds of resistances of the pprI mutant. In addition, recombination rate analysis and Western blotting results showed that mutation of D. radiodurans recX could up-regulate the basal expression levels of D. radiodurans RecA and PprA under normal conditions. Taken together, these results suggest that PprI regulates the induced expression of RecA and PprA via antagonizing RecX function in this bacterium.
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