CHAPTER 5 SUMMARY AND CONCLUSION. 188 P a g e

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1 CHAPTER 5 SUMMARY AND CONCLUSION 188 P a g e

2 Deinococcus radiodurans R1 exhibits an extraordinary tolerance to various abiotic stresses including radiations and desiccation. The amazing radioresistance of this bacterium is believed to be largely contributed by an efficient DSB repair and a strong oxidative stress tolerance mechanism. D. radiodurans exposed with radiation, shows gamma radiation mediated differential expression of a large number of genes at transcriptional levels and protein turnover with a unique process of protein recycling. The molecular mechanisms underlying the gamma radiation effects on transcription and protein turnover are not known in this bacterium. However, unlike E. coli and many other bacteria, where DNA damage induced genes expression have been implicated to SOS response, however existence of SOS repair in Deinococcus radiodurans has been ruled out. The possibility of an alternate mechanism to gamma radiation response could be envisaged in this bacterium. Interestingly, the genome of D. radiodurans R1 encodes a large number of hypothetical proteins, proteins known for recombination repair in other bacteria except RecBC enzymes. In addition, it also encodes numbers of eukaryotic type Ser/Thr protein kinases (estpk), stress response regulator and sensor proteins including histidine kinases and key regulatory enzyme named as PQQ synthase (pqqe) involved in biosynthesis of pyrroloquinoline quinone (PQQ), a well characterized antioxidant both in mammalian system and bacteria. Also PQQ was shown earlier, supporting the oxidative stress tolerance in E. coli and having role as an activity inducer of Ser/Thr protein kinase that has a role in radiation resistance in E. coli. This study focused on identification of PQQ inducible putative Ser/Thr kinase from D. radiodurans and further understanding the mechanism underlying this kinase role in gamma radiation resistance and DSB repair of this bacterium. Since, PQQ was indicated as an inducer of STPK, could support the oxidative stress tolerance and the E. coli cells expressing pqqe from D. 189 P a g e

3 radiodurans R1 became mineral phosphate solubilization positive, a phenotype regulated by PQQ in bacteria. These findings have provided enough indication on the functional status of pqqe albeit using E. coli as a heterologous system. In order to understand this gene role in D. radiodurans, the pqqe was disrupted with antibiotic marker in the genome of this bacterium and mutant was characterized for its response to different DNA damaging agents including gamma radiation. Mutant showed a severe loss of gamma radiation resistance and hydrogen peroxide but no effect was observed on UVC resistance, as compared to wild type cells. These cells also show impairment in DSB repair. These findings suggest a role of PQQ in gamma radiation resistance and DSB repair in this bacterium. In order to investigate the molecular basis of PQQ action in gamma resistance and DSB repair, the five ORFs (DR_0503, DR_0766, DR_1769, DR_2518 and DR_C0015) encoding polypeptides having multiple beta propeller motifs characterized for PQQ interaction in dehydrogenases, were individually deleted from D. radiodurans genome. Deletion mutants were checked for gamma radiation resistance. Interestingly, dr2518 deletion ( dr2518) made D. radiodurans cells hypersensitive to the effects of different DNA damaging agents like gamma, UVC, hydrogen peroxide, mitomycin C and 5% desiccation. Other deletions did not show any change in wild type response to gamma radiation and effects of other agents were not checked on remaining mutants. This indicated that DR2518, which is also annotated as a putative STPK in the genome of D. radiodurans, has a role in DNA damage tolerance of this bacterium. The possibility that PQQ could have functioned through DR2518 was further strengthened as double mutant ( dr2518 pqqe:cat) did not show additive effect and levels of gamma resistance were similar to single dr2518 mutant. This might suggest that both pqqe and dr2518 function through common pathway in conferring radioresistance to D. radiodurans. The functional interaction of PQQ with DR2518 was further studied at protein levels. 190 P a g e

4 Recombinant DR2518 was made and the purified protein showed autophosphorylation irrespective of PQQ presence but this activity of DR2518 was enhanced in presence of PQQ in solution. DR2518 was further checked for its in vivo phosphorylation both in wild type and pqqe:cat mutant cells. DR2518 autophosphorylation was although observed in both cells; its stimulation in response to gamma radiation was not observed in pqqe mutant. Transcriptome data showed the stimulation of both dr2518 and pqqe transcription in response to gamma radiation. These results indicated that DR2518 is phosphoprotein irrespective of PQQ presence and provide evidence although indirectly to suggest that gamma radiation induces the levels of both DR2518 and PQQ, which in turned stimulate the activity of DR2518 kinase in vivo. Thus the roles of both PQQ and DR2518 kinase in gamma radiation resistance and DSB repair are suggested. Structure-function studies using bioinformatic tools, the catalytic cleft and activation loop of DR2518 were defined and using site directed mutagenesis approach the amino acids residues responsible for the kinase activity of DR2518 have been identified. The K42A mutant of DR2518 was nil in kinase activity also failed to complement the function of DR2518 in dr2518 mutant suggesting that kinase activity of DR2518 is per se required for the contribution of DR2518 in radioresistance of this bacterium. Using bioinformatic approaches, the proteins having putative phosphorylation motifs were scanned in the proteomes of D. radiodurans. A large number of proteins were found having phospho-motifs characterized for proteins as substrate of estpk of mycobacterial kinases. Some of these proteins were checked for phosphorylation ability by wild type, different mutated derivative and only by kinase domain of DR2518. Interestingly, we observed that both full lengths DR2518 could phosphorylate many including PprA (a pleiotropic protein involved in DNA repair) but not K42A mutant. PprA phosphorylation by full-length DR2518 was stimulated but not by protein kinase domain missing 191 P a g e

5 C-terminal sensory beta propeller repeats. These results suggest DR2518 could phosphorylate deinococcal-proteins in vitro; its ability to discriminate from the promiscuity is most like lying with C-terminal domain of DR2518 kinase. Nonetheless, these findings strongly supported the involvement of an estpk of D. radiodurans in its radiation resistance and DSB repair by phosphorylating the DNA metabolic proteins including PprA, a known DNA repair protein in this bacterium. Taking PprA as a representative candidate, we assessed the functional relevance of DNA metabolic proteins phosphorylation. Recombinant PprA was purified and phosphorylation was achieved in presence of DR2518 incubated with ATP. PprA was known for its DNA binding activity, its role in stimulation of DNA end joining activity of DNA ligases and DNA protection from exonuclease III degradation. Phosphorylated PprA (P-PprA) was compared with unphosphorylated form of PprA (PprA), for these three well-characterized roles in vitro and significance of P-PprA in radiation resistance using functional complementation in ppra deletion mutant. P-PprA stimulated its dsdna binding property by nearly 4 folds and DNA end joining activity of T4 DNA was switched from intramolecular ligation to intermolecular ligation in presence of P-PprA. The exonuclease III protection was however, reduced in presence of P-PprA. These results suggested that PprA phosphorylation alters its in vitro functions. The phosphoablative T72A mutant of PprA showed significantly lesser phosphorylation by DR2518 and accordingly low levels of gamma radiation resistance complementation by phosphomimitic T72D in ppra deletion mutant. This suggested that phosphorylation of PprA by DR2518 at least under this study, could change the functional characteristics of this protein both in vitro and in vivo. Although, the involvement of protein phosphorylation in regulation of gene expression is yet to be established, the findings from this study has laid down the possibility of eukaryotic type STPK roles in gamma radiation resistance 192 P a g e

6 and modulation of DNA metabolic proteins functions e.g. PprA could be suggested. The phopshorylation mediated higher turnover of existing proteins in absence of their de novo synthesis during early post irradiation recover period may be required to cope up the greater demand of their functions under PIR and DR2518 or other possible kinases contribute to this requirement cannot be ruled out. Further the bacterium like D. radiodurans, which lacks classical DNA damage response mechanisms like SOS and extremely resistant to DNA damaging agents, the STPK mediated gamma radiation response mechanisms might provide the basis for further studies leading to the discovery of alternate DNA damage response in this bacterium. Conclusions 1. PQQ roles in radiation resistance and DSB repair of Deinococcus radiodurans R1 have been demonstrated. Evidences were provided to suggest that PQQ functions as an inducer of a radiation responsive estpk protein kinase DR2518 in D. radiodurans and thus the role(s) of protein phosphorylation in radiation resistance and DSB repair in this prokaryote could be hypothesized. 2. DR2518 was characterized as eukaryotic type Serine / Threonine quinoprotein kinase (estpk). Stimulation of DR2518 protein kinase activity by PQQ and no stimulation of only N-terminal kinase domain of DR2518 by PQQ indicated the role of C-terminal domain containing beta propeller repeats (PQQ binding motifs) as a regulatory domain. Although, the molecular basis of DNA ends stimulation is not known, the possibility of this protein forming DNA interaction motifs upon dimerization may be speculated and required further investigation. 193 P a g e

7 3. Lysine 42 (K42) is catalytically important residue for the kinase activity of DR2518, and Threonine-169 (T169) was found to be a phosphoacceptor site in activation loop of this kinase. 4. Protein kinase activity of DR2518 kinase is per se required for gamma radiation resistance in D. radiodurans. 5. DR2518 kinase could phosphorylate various proteins (RecA, PprA, DR0012 (putative chromosome I partitioning protein) and DRB0002 (plasmid partitioning protein). Phosphorylation of PprA has a role in its functional efficiency both in vitro and in vivo. 6. Thus, the present study reports for the first time the role of any eukaryotic type STPK in radiation resistance and DSB repair in a prokaryote, and provided a plausible basis to its function in radioresistance of D. radiodurans. Future prospective: This thesis is the first report studying the roles of STPKs in bacterial response to gamma radiation and oxidative stress. It has brought forth the direct or indirect answers to many interesting questions that are very closely related to gamma radiation response in Deinococcus radiodurans. On the other hand, this study has generated a number of intriguing facts required for investigation to complete the pathway (s) regulating DNA damage response in this bacterium. Some of the worth pursuing on priority would be as follows. 1. How does phopshorylation of DNA metabolic proteins associated with DNA repair and recombination contribute to gamma radiation resistance in D. radiodurans? 2. Does DR2518 sit at crossroad of oxidative stress and gamma radiation induced DNA damage inducible signaling processes? 194 P a g e

8 3. Why the gamma radiation inducible autophosphorylation of DR2518 was not observed in pqqe mutant would be worth investigating? 4. What are the roles of other putative quinoproteins in D. radiodurans? 195 P a g e