Biochemical Characterization And Mutational Analysis Of RecQ And Its Related Gene In Deincoccus Radiodurans

Deinococcus raidoudrans can survive extreme DNA damage caused by ionizing radiation and other DNA damage agents at levels that would otherwise kill Escherichia coli and other related organisms. The survive of D. raidoudrans in such extreme conditions is due to its extraordinary DNA repair ability, especially in the reconstruction of dsDNA breaks induced by ionizing radiation. The incredible DNA repair capacity makes D. radiodurans an excellent model system. However, current studies have only touched the surface, and much is still to be learned from D. radiodurans.The RecQ family of DNA helicases performs essential functions in the maintenance of genomic stability in all organisms. In D. radiodurans, DR1289 is a special member of RecQ family with unique arrangement of three tandem HRDC domains in the C-terminus. Moreover, single stranded binding protein (SSB) that interacted with RecQ plays crucial in all kind of DNA metabolic processes includeing DNA replication, recombination and repair. In contrast to other bacterial SSB proteins, which are functionally active in homotetramers, Deinococcus SSB-like proteins are homodimers in nature and contain two OB fold per monomer. In this study, we investigated biochemical characterization and mutational pattern of recQ and its related gene in D. radiodurans. The results were listed as below:1. RecQ mutant was constructed at first. A dr1289 mutant is hypersensitive toγ-irradiation, UV, H2O2 and mitomycin C. 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 complete DNA damage resistance. We also found that the helicase domain is necessary for the unwinding and ATPase activity, and the three tandem HRDC domains increase the efficiency of these activities.2. Four truncated variants of DrSSB proteins were generated based on their crystal structures. Gel filtration showed that DrSSB, DSCT and DSCC were homodimers, and DSN and DSNC were monomers. The gel filtration analysis supported the hypothesis that the N-terminal domain played a predominant role in dimerisation. Biochemical characterization was used to determine the role of each OB fold in DNA binding, by electrophoretic mobility shift assay (EMSA) and fluorescence resonance energy transfer analysis (FRET).4. A four-gene mutant strain was created by constructing recQ knockout mutant in uvrAl, uvrA2, and uvsE knockout backgrounds. Using the rpoB/Rif system, we measured the mutation frequencies and rates in wild type, recQ (MQ), uvsE uvrAl uvrA2 (TNK006), and uvsE uvrAl uvrA2 recQ (TQ). We then isolated Rif mutants of these strains and sequenced the rpoB gene. The results indicate that recQ is involved in the ultraviolet damage repair pathway via the interaction between recQ and uvrAl, uvrA2, and uvsE in D. radiodurans.5. The mutation frequencies and rates of D. radiodurans were measured at a wide concentration range from 5 to 50μg/ml of rifampin. We found that the mutation rate of the bacterium in the presence of 5μg/ml of rifampin was significantly higher than in the presence of 25μg/ml and 50μg/ml rifampin. Rifampin had concentration-denpendent effect not only on the mutation rate, but also on the mutation spectrum. It is speculated that D. radiodurans mainly prevents base substitution mutation at low concentration of rifampin, since ROS induced mainly oxidative base damage6. DNA microarrays and biochemical assays were used to identify the molecular details of how the Rif mutation in the P-subunit led to changes in growth rate via altered regulation of multiple genes in D. radiodurans. The results provided a molecular insight into the mechanism of Rif mutation in theβ-subunit on altering the regulation of multiple genes.Taken together, the three HRDC domains of RecQ helicase is important not only for radiation resistance, but also for helicase activity and ATPase activity of RecQ. SSB that interacted with RecQ needed the interaction between two OB folds. Mutational analysis suggested that recQ is involved in the ultraviolet damage repair pathway. Rifampin had concentration-denpendent effect not only on the mutation rate, but also on the mutation spectrum in D. radiodurans. The microarray results provided a insight into the mechanism that Rif mutation inβsubunit alter the regulation of multiple genes.