Study On The Serine/Threonine Phosphokinase Family Genes In Deinococcus Radiodurans

Reversible phosphorylation on serine, threonine and tyrosine of protein change the steric Hindrance, polarity and stability of amino acid, and give protein different activity, location, stability, and interaction. Phosphorylation functions as a regulator in cell cycle, cell differentiation, stress response and so on. Deinococcus radiodurans is one of the most radio-resistant organisms. It has an extreme resistance to the stress of y radiation, UV radiation and hydrogen peroxide. D. radiodurans can survive a5kGy ionizing radiation with no lethality. Efficient DNA repair mechanism, a variety of antioxidant and rapid DNA damage mechanisms contribute to the extreme radio-resistance of D. radiodurans. Most researches on D. radiodurans focus on DNA repair and antioxidation, however, the knowledge on the rapid DNA damage response mechanisms is quite limited. Moreover, the roles of protein phophorylation in post-irradiation recovery (PIR) are still poor known.D. radiodurans genome contains over20genes encoding serine/threonine phosphorlation kinases. Here, we selected ten of them to investigate their roles in the PIR, including DR0058, DR0534, DR1213, DR1243, DR1851, DR2108, DR2518, DR2546, DRA0332and DRA0334. We first constructed the knockout mutant strains of the ten genes and then investigated the survival rates of these mutant and wildtype strains under the stress of DNA damage agents. We found that the mutation of seven genes (dr0058, dr0534, dr1213, dr1851, dr2108, dra0332, dra0334) did not effect on the radio-resistance of D. radiodurans, indicating that these seven genes have no significant contribution to ionizing radiation resistance and may not involve in regulation of DNA repair mechanisms during PIR. However, mutation of dr1243and dr2518caused a obvious growth delay of D. radiodurans. Moreover, the mutation of the three protein kinase gene (dr1243, dr2518, dr2546) led to significant decrease on resistance to y radiation, UV radiation and hydrogen peroxide. The lack of dr2518lead to more sensitivity of D. radiodurans to y radiation and UV radiation, which is similar to the results reported previously. The deletion of dr1243in D. radiodurans caused10times reduction of survival rate with treatment of y radiation and hydrogen peroxide at high dose, significantly more sensitive than the dr2546gene deletion. Data indicates that the three kinases (DR1243, DR2518, DR2546), especially DR1243, contribute to the radio-resistance and the recover mechanisms after irradiation.Based on the contribution of the three kinases, especially DR1243, to the resistance of D. radiodurans, we further explored how DR1243kinase works in the cell. We use gene chip technology to detect the change of RNA transcript levels in dr1243mutant strain and to study the regulated gene by dr1243in vivo. Results show that deletion of dr1243led to down-regulation of a large number of genes, including RNA ligase DRB0094and iron regulatory protein Fur (DR0865), which indicate that dr1243may indirectly regulate the RNA repair, Fe levels in vivo, the concentration of free radicals, Mn/Fe ratio and other factors affecting cell resistance. We give a preliminary analysis on how dr1243regulate cell resistance in vivo.In summary, seven phosphorlation kinases (DR0058, DR0534, DR1213, DR1851, DR2108, DRA0332, DRA0334) contribute little to the radio-resistance of D. radiodurans and three kinases (DR1243, DR2518, DR2546) were evidenced their crucial roles in the extreme resistance to y radiation, UV radiation and hydrogen peroxide of D. radiodurans. The further study on dr1243showed that the kinase regulates the RNA repair, Fe levels in cells, the concentration of free radicals, Mn/Fe ratio and other factors affecting the cell resistance. Taken together, protein kinases are evidenced their involvments in the PIR of D. radiodurans.