Estudo do papel da homeostase do cálcio na reparação de danos no DNA em diferentes linhagens de Saccharomyces cerevisiae

Abstract

Calcium (Ca2+) has been reported as an important cellular ion. The changes in the intracellular Ca2+ concentrations are related to: (i) the activation of major proteins related to DNA repair and apoptosis; (ii) induction of human diseases; and (iii) increase in cellular damages. Considering that the Ca2+ intracellular concentration is also responsible for the maintenance of cellular viability, the aim of this study was to proceed with a literature data mining to outline the major Ca2+ molecular mechanisms that can affect the cellular activities. This data mining was necessary to define further biological assays described in this work. In this sense, it was developed a new methodology called PDQT, which allows to evaluate the survival of different Saccharomyces cerevisiae strains deficient or proficient for determined proteins in a chronic exposure conditions in the presence of a genotoxic agent. This new method also allows to observe the citotoxic, citostatic, and cell cycle arrests effects induced by a genotoxic agent. Moreover, it was performed a Systems Biology analyses of the major S. cerevisiae Ca2+ homeostasis-associated proteins and it was found a direct association with proteins of the nucleotide excision repair (NER) pathway. The data gathered by Systems Biology analyses were used to construct yeast strains single and double mutants deficient for Ca2+ homeostasisassociated proteins, like Pmr1p, Cod1p, Por1p, and for the NER-associated protein Rad4p. The yeast strains obtained were challenged against the genotoxic agent 4-NQO and citotoxic, citostatic, and cell cycle arrest assays were performed. Taking into account the results, it was observed that Ca2+ homeostasis mechanisms affect the DNA repair pathways, specially the NER proteins like Rad4p. In this sense, both Cod1p and Pmr1p are the most important Ca2+ homeostasis proteins for the damage cellular response. In addition, these results propose that ER stress and the phosphorylation of cell cycle arrest-associated proteins can be the major metabolic processes after 4-NQO exposure. The results obtained by System Biology and gene ontology analyses proved that many biochemical pathways are related to Ca2+ homeostasis, which demonstrate the importance of the results obtained in this work and lead to other studies.