Metabolismo de DNA em Trypanosoma cruzi: caracterização dos genes RAD51 e DNA polimerase

Abstract

Trypanosoma cruzi is a parasite of the Kinetoplastida order, endemic in Latin America, where it causes Chagas disease in humans. An interesting aspect of its biology is its population structure, composed of strains with highly differential features. The T. cruzi genome is highly complex, rich in repetitive elements and containing a large number of multi-copy gene families. However, little is known about the mechanisms involved in the maintenance of such a complex genome and only few genes involved in DNA metabolism in this parasite have been characterized. Our work was intended to contribute to the understanding of this important aspect of the parasite biology, i.e., how genetic variability is created and, at the same time, how the integrity of the parasite´s genetic material is preserved. Two key genes involved with those processes were studied: Rad51, because of its crucial role in DNA recombination and DNA polymerase , which codes an enzyme of base excision repair. We have shown that T. cruzi Rad51 gene interferes with the recombination machinery of eukaryotic cells, causing an increase in the rate of these events in a mammalian cell line transfected with the T. cruzi gene. Furthermore, the Rad51 gene can be associated with the T. cruzi response to DNA lesions caused by radiation because exposition to this agent provokes an increase in Rad51 mRNA levels. The T. cruzi gene encoding DNA polymerase is able to complement bacteria cells deficient in DNA polymerase I when expressed as a fusion with the MBP gene. It is also able to perform in vivotranslesion synthesis in these bacteria. The DNA synthesis capability of the recombinant protein was confirmed in vitro using the MBP-DNA pol protein fusion, and it was shown to present a characteristic low fidelity synthesis. Using a non-radioactive assay developed by us, we tested the DNA pol enzymatic activity under different conditions. It was also possible to verify that the recombinant TcDNA pol is able to insert modified nucleotides such as azidothymine (AZT) triphosphate and didesoxinucleotide triphosphate in DNA, which is an enzymatic activity characteristic of the polymerase X family. Because treatment of T. cruzi epimastigote cultures with AZT results in parasite death, we inferred that TcDNA pol may be one of the enzymes responsible for incorporating this drug into the parasites genome. Here, we have characterized the Rad51 and DNA pol genes of T. cruzi contributing to better understand their roles in its DNA metabolism.