Regulatory Network And Functions Of Two Key RecQ Genes In Maintaining Genome Stability Of Deinococcus Radiodurans

The bacterium Deinococcus radiodurans is extremely resistant to ionizing radiation, ultraviolet, hydrogen peroxide and many other agents that damage DNA. Studies show that this resistance is due to D. radiodurans's extremely proficient and accurate DNA repair process. After the release of genome sequence of D. radiodurans wild type strain R1 in 1999, the structure characteristics, molecular defense systems and the biochemical mechanisms of many key repair genes, such as recA,ssb,pprI,pprA are making great progress. But the accurate regulatory network of DNA repair in Deinococcus radiodurans is still remaining unknown in large part.As a member of SF1 superfamily, the RecQ helicases are highly conserved in evolution and are required for maintaining genome stability in all organisms. D. radiodurans encodes two recQ genes with unusual domain, DR1289 and DR2444, whose functions, however, remain obscure currently. In this paper, the biochemical mechanism and regulatory network of the two genes were studied thoroughly. The main work and its results are appended following:1. Using bioinformatic methods, the loci of DR1289 and DR2444 in the chromosome were localized. Sequence alignment of different domains, biochemical function prediction and construction of phylogenetic tree were performed. Results obtained from all above studies suggest that DR1289 has a close phylogenetic relationship with conserved RecQ family members from bacteria to eukaryotic organisms, whereas DR2444 has distant relationship with RecQ family.2. A fusion DNA fragment carrying kanamycin resistance gene with the D. radiodurans groEL promoter, chloramphenicol resistance gene with KAT promoter was cloned by PCR amplification and inserted into the recQ locus in the genome of the wild-type strain R1. Three resulting recg-deficient strains, designated â–³DR1289,â–³DR2444 andâ–³recQ (double mutation), were constructed. Results show thatâ–³DR1289 andâ–³recQ were very sensitive to ionizing radiation and H₂O₂, whileâ–³DR2444 was not. The phenotype ofâ–³DR1289 was similar to many RecQ helicase mutants. Therefore, it was presumed that DR1289 was the necessary gene in maintaining the extreme resistance to DNA damaging agents, whereas DR2444 was not. Further research based on genetic and biochemical approaches should help togain a better understanding of the genes involved in DNA repair.3. Some phenotypes of mutantâ–³DR1289 were characterized. The mutant strainâ–³DR1289 was sensitive to mitomycine C, hydrogen peroxide and UV. Interestingly, when the dosage of gamma radiation up to 2 kilograys, the radiation resistance of the mutant decreased remarkably compared to that of the wild type R1. After the deletion of DR1289, growth rate of the mutant was significantly slower. In addition, results of pulsed-field gel electrophoresis (PFGE) showed that the genome stability was destroyed in DR1289 mutant. After 20 mM H₂O₂ treatment for 30 minutes, genome mapping of the mutant presented in PFGE was very similar to R1 treated with high dose gamma irradiation and prolonged desiccation. By complementing the DR1289 mutant with various domains of DR1289 in vivo, we have determined that the helicase and all three HRDC domains are indispensable for DNA damage resistance.4. The DR1289 protein and its variant proteins were expressed by E. coli expression system and then purified. Using a continuous fluorescent dye-displacement assay, we investigated the optimal conditions for DR1289 unwinding function at various concentrations of ATP and metal ions, indicating that the helicase activity is comparable to that observed of E. coli RecQ. SSB protein servers as a stimulatory factor of the unwinding by DR1289. The unwinding rate of DR1289 increases by addition of D. radiodurans SSB protein, DrSSB has a species-specific effect on DR1289. We also found that the helicase domain is necessary for the ATPase activity and that the three tandem HRDC domains increase the efficiency of these activities. Based on these data, we propose that the C-teiminus of DR1289 has evolved in D. radiodurans to confront the types and amounts of DNA damage caused by this organism's extreme environment.5. Global transcriptome profiles response to DR1289 deletion showed that 9.1% of genome changed at least 2-fold in DR1289 mutant strain, such as iron homeostasis, oxidative related genes, and cell division related genes. Furthermore, under 20mM H₂O₂ stress, the profile expression patterns of many genes (e.g., recA, pprA, ddrA, DR0003, DR0070) were similar to acute gamma-irradiation and prolonged desiccation. Western blotting results also demonstrated that RecA was up-regulated in DR1289 mutant, indicating that there were vast DNA damages in vivo. Mn (II) accumulation (with low Fe) facilitates resistance in D. radiodurans, but in DR1289 mutant, the intracellular level of Fe is higher, Mn is lower, which decreased the Mn/Fe ratio significantly, whereas free iron in DR1289 mutant in only fifty percent of wild type by EPR experiment. Taken together, DR1289 is a key gene in maintaining the genome stability and contributing the extraordinary ability to D. radiodurans.6. To further unlocking the regulatory mechanism of DR1289 in D. radiodurans, ProteomeLab PF 2D protein quick separation method has been developed for differential display of proteins from cell lysates and applied to a comparison of protein expression between wild type and DR1289 mutant in 20 mM H₂O₂ stress.Taken together, among the two recQ genes in D. radiodurans, DR2444 is not the necessary gene to extreme resistance, on the contrary, DR1289 acts as a key gene with multiple roles in vivo and in vitro, which is required for maintaining genome stability, cellular homeostasis and interaction with many important genes.