Role of Trypanosoma cruzi nucleoside diphosphate kinase 1 in DNA-damage responses

NME23/NDPK proteins are well conserved proteins found in all living organism. Besides their catalytic activity of nucleoside diphosphate kinase (NDPK) they are considered multifunctional, which were first characterized as non-metastatic proteins in mammalian cells. Later, increasing evidences placed NME/NDPK as proteins involved in DNA stability such as gene regulation and DNA-repair. TcNDPK1 is the canonical NDPK isoform present in the parasite Trypanosoma cruzi, orthologous to NME23-H1/H2 which has been shown to have in vitro nuclease activity and DNA-binding properties. In the present study we investigate the role of TcNDPK1 in DNA-damage responses using heterologous gene expression systems and over-expression in epimastigote cells. We found that different strains of bacteria, WT and ndk-mutants, expressing the enzyme decreased about 5 fold and 18 fold the spontaneous mutation rate, respectively. In addition, yeasts lacking the endogenous gene YNK1 (YNK1-) and expressing TcNDPK1, were significantly more resistant to different concentrations of hydrogen peroxide and were less sensible to UV radiation than controls. Parasites over-expressing TcNDPK1 were able to withstand different genotoxic stresses caused by hydrogen peroxide, phleomycin and hidroxyurea. In addition, under oxidative damage, TcNDPK1 over-expressing parasites presented lesser genomic damage and augmented levels of poly(ADP)ribose and poly(ADP)ribose polymerase, an enzyme involved in DNA repair. These results strongly suggest that TcNDPK1 is involved in the maintenance of parasite genomic-DNA integrity, thus, giving rise to a novel function.


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Nucleoside diphosphate kinases (NDPK) are ubiquitous and well conserved enzymes 50 whose canonical and first discovered function is to maintain the intracellular di-and tri-51 phosphate nucleotide homeostasis by catalysing their interconversion [1]. In addition,

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NDPK proteins are considered multifunctional enzymes since they have been shown to 53 be involved in diverse physiological and pathological processes. Human NDPKs, 54 comprised in the NME (NME23/NDPK) family, are by far the best studied NDPKs 55 because of their prime role as metastasis suppressors [2]. The different isoforms have 56 been found to be distributed all along the intracellular compartments, from cytoplasm to 57 mitochondria, membrane and nucleus [3,4]. Nuclear functions of NME proteins are 58 currently being investigated in order to better understand the mechanisms underlying 59 its non-metastatic function. NME-H1 and NMEH-2 have been identified as potential 60 transcription factors that regulate gene transcription through their DNA-binding 61 activities [5][6][7][8][9]. In addition, NME-H1, NME-H5, NME-H7, and NME-H8 also exhibit a 3′-

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there are increasing studies confirming a possible role for NME-H1 in the repair of DNA stresses as is showed in figure 1 (Fig 1), being significantly more resistant to 5, 10, 25 238 and 50 mM hydrogen peroxide after 24 h of treatment and to 4.25 and 8.5 J/cm 2 UV 239 exposure than controls (Fig 1C). GFP. In addition, the transgenic lines had similar culture growth and duplication rates,

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reaching same densities at stationary phase (Fig 2A). shown in figure 2 (Fig 2). Markedly, at H 2 O 2 100 µM 100 % of N1 parasites survived to 265 the treatment while GFP parasites grew only 20 %; in addition, H 2 O 2 125 µM was 100% 266 lethal to GFP parasites and N1 population persisted reaching 50% of growth (Fig 2 B) able to endure about 24% more than GFP parasites at HU 10 mM and 20 mM (Fig 2D).  fragmentation was observed, so parasites were then exposed briefer times to 3 mM 290 H 2 O 2 (0, 5, 10, 20 and 30 min). Accordingly with the results obtained above, N1 291 parasites presented slightly less DNA damage than control parasites, as can be seen 292 in figure 3 (Fig 3A and 3B).

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GFP parasites were used as control.

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Different DNA damage responses are activated under these genotoxic conditions. One examine the activation of TcPARP in N1 and control populations, parasites were 303 exposed to H 2 O 2 3 mM at different times (0, 5, 10, 20 and 30 min) and PARylation was 304 evaluated by western blot (Fig 4A). In both parasites the level of modified proteins 305 increased with damage until 20 min, but N1 parasites presented more intensity than 306 control parasites and, remarkable, they maintained the elevated levels at 30 min 307 against GFP parasites that showed an important decreased of about 46% at the same time ( Fig 4B). The levels of TcPARP were also evaluated ( Fig 4C) and in accordance 309 with PARylation, the levels augmented with time exposure and in N1 parasites 310 continued increasing at 20 min keeping them high at 30 min, whereas control parasites 311 showed an abrupt decreased being almost undetectable at the mentioned times (Fig   312  4D). In addition, at 5 and 10 min, induction of expression was greater in N1 parasites, 313 reaching about 6 to 10 fold respect to 0 min against 1.5 and 1.7 fold of GFP parasites 314 (Fig 4D).

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Previous experiments carried out in our laboratory threw the presence of NDPK activity 322 in nuclear fractions (Miranda et al, unpublished) and, in addition, we determine that 323 TcNDPK1 has a cytosolic localization, enriched mainly around the nucleus [20,21] 324 Furthermore, the orthologous gene of TcNDPK1 in T. brucei codifies a NDPK that was 325 reported as nuclear enzyme [23]. In order to evaluate in detail the subcellular 326 localization of TcNDPK1, immunofluorescences were carried out in these transgenic 327 parasites. As is shown in figure 5, in addition to be cytosolic, TcNDPK1 showed 328 marked nuclear and peri-nuclear localization, very similar to its T. brucei counterpart 329 (Fig 5, for details, please see [23]). In addition, phleomycin and H 2 O 2 treatment 330 produced a slight delocalization of TcNDPK1 out of the peri-nuclear region (Fig 5).