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| Author(s): |
Jeziel Dener Damasceno
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2013-02-06 |
| Examining board members: |
Luiz Ricardo Orsini Tosi;
Angela Kaysel Cruz;
Juliana Lopes Rangel Fietto;
Carlos Frederico Martins Menck;
Sergio Schenkman
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| Advisor: | Luiz Ricardo Orsini Tosi |
| Abstract | |
Genome stability is a essential condition for survival and proper functioning of living organisms. However, a broad range of elements may lead to DNA damage. For instance, about 104 DNA lesions may be inflicted upon any given mammalian cell everyday. In order to maintain the genome integrity and circumvent the deleterious effects of these lesions, a molecular machinery composed of proteins specialized in detecting and repairing DNA damage has been selected in evolution. Defects of the proteins that constitute such machineries may result not only in a high mutation rate, but also in breaks in the DNA structure that can mediate gene amplification as observed in cancer cells. In an apparent opposition to such requirement for stability as an essential condition to life, the protozoan Leishmania presents a highly malleable genome and explores genome amplification as a survival and adaptation tool. Despite of the fact that the Leishmania genome plasticity can be easily demonstrated, the precise mechanisms that coordinate the molecular machineries involved in the detection and signaling of DNA damage, and in the regulation of gene amplification is still largely unknown. In order to contribute to a better understanding of these processes, we identified and studied the Leishmania major proteins that are homologues of those proteins that compose the 9-1-1 complex (Rad9-Hus1-Rad1). The proteins LmHus1 and LmRad9 present a high structural divergence when compared to its homologues from other eukaryotes and no obvious homologue of Rad1 was identified in the parasite genome. Phylogeny analysis indicated that LmHus1 and LmRad9 are closely related to heterotrimeric complexes involved in the detection of DNA damage. In accordance to that, our experiments demonstrated that altered levels of these proteins interfere with the parasite ability to deal with genotoxic stress. Moreover, LmHus1 was localized to the parasite nucleus and is a required protein for normal parasite proliferation. Besides, we showed that decreased levels of LmHus1 compromise cell cycle regulation and the maintenance of telomeres. LmRad9 was also shown to be localized to the cell nucleus and its overexpression led to growth defects and affected the L. major response to genotoxic stress. We also observed that LmHus1 and LmRad9 interact with each other to for a protein complex that is responsive to DNA damage in vivo, which strongly suggested that the 9-1-1 complex was conserved in L. major. The structural peculiarities of these proteins indicate that the possible L. major 9-1-1 complex has a different architecture when compared to the complex found in higher eukaryotes. In addition to that, other proteins, such as LmRpa1, also present a marked structural divergence. Altogether, these findings suggest that the DNA damage signaling pathway involving the 9-1-1 complex and LmRpa1 in L. major, may present a peculiar mode of action. These observations may contribute to a better understanding not only of the evolution of the signaling pathway mediated by the 9-1-1 complex in eukaryotes, but also of the molecular basis of the genome plasticity and the gene amplification phenomenon. (AU) | |