Hypothetical proteins present during recovery phase of radiation resistant bacterium Deinococcus radiodurans are under purifying selection.

Deinococcus radiodurans has an unusual capacity to recover from intense doses of ionizing radiation. The DNA repair proteins of this organism play an important role in repairing the heavily damaged DNA by employing a novel mechanism of DNA double-strand break repair. An earlier report stated that genes of many of these repair proteins are under positive selection implying that these genes have a tendency to mutate, which in turn provides selective advantage to this bacterium. Several "hypothetical proteins" are also present during the recovery phase and some of them have also been shown for their roles in radiation resistance. Therefore, we tested the selection pressure on the genes encoding these poorly characterized proteins. Our results show that a number of "hypothetical proteins" present during the repair phase have structural adaptations compared to their orthologs and the genes encoding them as well as those for the DNA repair proteins present during this phase are under purifying selection. Evidence of purifying selection in these hypothetical proteins suggests that certain novel characteristics among these proteins are conserved and seem to be under functional constraints to perform important functions during recovery process after gamma radiation damage.

DR2417, a hypothetical protein characterized as a novel ß-CASP family nuclease in radiation resistant bacterium, Deinococcus radiodurans.

BACKGROUND: Deinococcus radiodurans survives extreme doses of radiations contributed by efficient DNA repair pathways. DR2417 (DncA) was detected separately both in a pool of nucleotide binding proteins and multiprotein complex isolated from cells undergoing DNA repair. SCOPE OF REVIEW: DR_2417m ORF was sequenced and amino acid sequence of DncA was search for structural similarities with other proteins and functional motifs. Recombinant DncA was characterized for its DNA metabolic functions in vitro and its role in radiation resistance. MAJOR CONCLUSIONS: Sequencing of DR_2417m did not show the reported frame shift at 996th nucleotide position of this gene. DncA showed similarities with ß-CASP family nucleases. Recombinant protein acted efficiently on dsDNA and showed an Mn2+ dependent 3'→5' exonuclease and ssDNA/dsDNA junction endonuclease activities while a very low level activity on RNA. The DNase activity of this protein was inhibited in presence of ATP. Its transcription was induced upon γ radiation exposure and a reduction in its copy number resulted in reduced growth rate and loss of γ radiation resistance in Deinococcus. CONCLUSION: Our results suggest that DncA was a novel nuclease of ß CASP family having a strong dsDNA end processing activity and it seems to be an essential gene required for both growth and γ radiation resistance of this bacterium. GENERAL SIGNIFICANCE: Traditionally DncA should have shown both DNase and RNase functions as other members of ß CASP family nucleases. A strong DNase and poor RNase activity possibly made it functionally significant in the radioresistance of D. radiodurans, which would be worth investigating independently.

Characterization of DRA0282 from Deinococcus radiodurans for its role in bacterial resistance to DNA damage.

DRA0282, a hypothetical protein, was found in a pool of nucleotide-binding proteins in Deinococcus radiodurans cells recovering from gamma radiation stress. This pool exhibited an unusual inhibition of nuclease activity by ATP. The N terminus of DRA0282 showed similarity to human Ku80 homologues, while the C terminus showed no similarities to known proteins. The recombinant protein required Mn(2+) for its interaction with DNA and protected dsDNA from exonuclease III degradation. The binding of the protein to supercoiled DNA with a K(d) of ~2.93 nM was nearly 20-fold stronger than its binding to ssDNA and nearly 67-fold stronger than its binding to linear dsDNA. Escherichia coli cells expressing DRA0282 showed a RecA-dependent enhancement of UV and gamma radiation tolerance. The ΔdrA0282 mutant of D. radiodurans showed a dose-dependent response to gamma radiation. At 14 kGy, the ΔdrA0282 mutant showed nearly 10-fold less survival, while at this dose both pprA : : catΔdrA0282 and pprA : : cat mutants were nearly 100-fold more sensitive than the wild-type. These results suggested that DRA0282 is a DNA-binding protein with a preference for superhelical DNA, and that it plays a role in bacterial resistance to DNA damage through a pathway in which PprA perhaps plays a dominant role in D. radiodurans.