Reparo de DNA em dois patógenos humanos: caracterização do gene IMP4 de Schistosoma mansini e estudos acerca do MMR, Sistema GO e taxa de mutação em Trypanosoma cruzi

Abstract

Cells have evolved a complex set of mechanisms to appropriately respond to genotoxic damage, ensuring genomic stability. Such responses retain mutation rates in an acceptable level, through a fine-tuning balance between genome maintenance and generation of genetic variability. In this work, we studied some features of the DNA repair machinery of Schistosoma mansoni and Trypanosoma cruzi, the causative agents of schistosomiasis and Chagas disease in Brazil. Both parasites need to adapt to distinct environments, and are consequently exposed to various DNA damaging agents. Using a functional heterologue complementation strategy, we have isolated a S. mansoni cDNA that complements Escherichia coli mutants defective in the DNA base excision repair (BER) pathway. This cDNA has sequence homology to a gene involved in the RNA metabolism pathway, the ScIMP4 gene of Saccharomyces cerevisiae. To establish whether the S. mansoni cDNA could complement yeast mutants ScIMP4-defective, we constructed a yeast haploid strain containing a truncated Imp4p gene which shows MMS sensitivity. The functional homology between the ScIMP4 gene and the cDNA from S. mansoni was verified by partial complementation of the mutant yeast with the worms gene. As a second goal, we studied T. cruzi DNA repair mechanisms possibly involved in the generation of genetic variability in this protozoan parasite. We have previously demonstrated that polymorphisms in the TcMSH2 locus, which encodes a key component of the mismatch repair (MMR), result in three different protein isoforms present in the T. cruzi population. Using cisplatin, MNNG and H2O2 in vivo assays performed with cadmium, which in sub lethal concentrations inhibits specifically the MMR, we provided evidences that TcMSH2A-expressing parasites have increased MMR activity when compared to parasite strains expressing TcMSH2C isoforms. To better understand the mechanisms involved in the oxidative DNA damage response in T. cruzi we characterized the putative OGG1 ortologue in the parasite and showed that (i) TcOgg1A expression is toxic in bacteria and (ii) stably TcOgg1A transfected parasites have increased H2O2 sensitivity. Additionally, a search for candidates for a functional homologue of E. coli MutT in T. cruzi using the Nudix Box was attempted. We`ve found that expression of Ndx1 or Ndx4 was unable to suppress the high spontaneous mutagenesis of E. coli mutT-deficient strain and that EcMutT overexpressing parasites showed increased survival and decreased 8oxoG levels after H2O2 treatment. Finally, we developed a methodology to determine the mutation rate in T. cruzi. The assay is performed in semisolid medium plates and is based on the selection of clones that spontaneously reverts the neomycin-sensitive phenotype caused by a single base substitution in the neomycin resistance gene (neo) inserted into the parasite genome. Our preliminary results indicate that this experimental approach is functional since (i) clones that grew in neomycin containing plates have the mutation repaired as shown by DNA sequence, (ii) a proportionally higher number of clones able to grow in the presence of neomycin was observed after long-time culture in liquid medium and in cultures that were treated with a mutagenic agent. This methodology is now in place in our laboratory to be used to determine the mutation rate in distinct T. cruzi strains, as well as analyzing the effect of different mutagens in this parasite.