BACKGROUND NME23/NDPKs are well conserved proteins found in all living organisms. In addition to being nucleoside diphosphate kinases (NDPK), they are multifunctional enzymes involved in different processes such as DNA stability, gene regulation and DNA repair among others. TcNDPK1 is the canonical NDPK isoform present in Trypanosoma cruzi, which has nuclease activity and DNA-binding properties in vitro. OBJECTIVES In the present study we explored the role of TcNDPK1 in DNA damage responses. METHODS TcNDPK1 was expressed in mutant bacteria and yeasts and over-expressed in epimastigotes. Mutation frequencies, tolerance to genotoxic agents and activity of DNA repair enzymes were evaluated. FINDINGS Bacteria decreased about 15-folds the spontaneous mutation rate and yeasts were more resistant to hydrogen peroxide and to UV radiation than controls. Parasites overexpressing TcNDPK1 were able to withstand genotoxic stresses caused by hydrogen peroxide, phleomycin and hidroxyurea. They also presented less genomic damage and augmented levels of poly(ADP) ribose and poly(ADP)ribose polymerase, an enzyme involved in DNA repair. MAIN CONCLUSION These results strongly suggest a novel function for TcNDPK1; its involvement in the maintenance of parasite's genome integrity.
24NME23/NDPK proteins are well conserved proteins found in all living organism. 25 Besides their catalytic activity of nucleoside diphosphate kinase (NDPK) they are 26 considered multifunctional, which were first characterized as non-metastatic proteins in 27 mammalian cells. Later, increasing evidences placed NME/NDPK as proteins involved 28 in DNA stability such as gene regulation and DNA-repair. TcNDPK1 is the canonical 29 NDPK isoform present in the parasite Trypanosoma cruzi, orthologous to NME23-30 H1/H2 which has been shown to have in vitro nuclease activity and DNA-binding 31 properties. In the present study we investigate the role of TcNDPK1 in DNA-damage 32 responses using heterologous gene expression systems and over-expression in 33 epimastigote cells. We found that different strains of bacteria, WT and ndk-mutants, 34 expressing the enzyme decreased about 5 fold and 18 fold the spontaneous mutation 35 rate, respectively. In addition, yeasts lacking the endogenous gene YNK1 (YNK1-) and 36 expressing TcNDPK1, were significantly more resistant to different concentrations of 37 hydrogen peroxide and were less sensible to UV radiation than controls. Parasites 38 over-expressing TcNDPK1 were able to withstand different genotoxic stresses caused 39 by hydrogen peroxide, phleomycin and hidroxyurea. In addition, under oxidative 40 damage, TcNDPK1 over-expressing parasites presented lesser genomic damage and 41 augmented levels of poly(ADP)ribose and poly(ADP)ribose polymerase, an enzyme 42 involved in DNA repair. These results strongly suggest that TcNDPK1 is involved in the 43 maintenance of parasite genomic-DNA integrity, thus, giving rise to a novel function. 44 45 46 47 65 different cell lines [10, 11]which is associated with an increased nuclear localization of 66 the proteins. 67 The DNA-related functions of NDPK proteins have also been investigated in different 68 organisms other than mammals, such as bacteria, plants, yeasts and protozoan 69 parasites. A plenty of reports commit NDPKs to DNA-processing activities because of 70 their interaction with DNA and their capacity to cleave it [12-17]. Furthermore, 71 accordingly to what was reported for human NME proteins, Deinococcus radiodurans 72 Ndk is up-regulated during gamma radiation stress [18] and the yeast homologue 73 YNK1 is required for repair of UV radiation-and etoposide-induced DNA damage [19].
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