| dc.description.abstract | Trypanosoma cruzi is a parasite of the order Kinetoplastida that causesChagas disease. A low level of allele divergence is observed in this organism. Although such high levels of homozygosity are not expected in an organism without sexual reproduction, this phenomenon may be promoted by genetic rearrangements during a recombination process. Recombination is also part of a pathway of double strand breakage repair, and the product of the Rad51 gene is one of the major proteins involved in this process. This enzyme has recombinase activity, whichresults in strand exchange between two DNA molecules. In this work, we have isolated and characterized the Rad51 gene from T. cruzi (TcRad51). A search in the EST database for sequences with homology with T. brucei Rad51 gene allowed us to design a set of primers used to clone the open reading frame for the Rad51 of T. cruzi by PCR. The nucleotide sequence was obtained and used to identify the protein sequence. Sequence comparisons in databases showed that TcRad51 protein hashomology with Rad51 of several organisms and with other recombinases. Also, some domains that are necessary to recombinase function were identified. Southern blot analysis showed that the TcRad51 is presented in two copies in the T. cruzi genome. Northern blot analyses indicated that one messenger RNA is expressed in all forms of the life cycle of this parasite. When expressed in wild type E. coli, the product ofthe TcRad51 gene was able to increase the mutation rate, probably by inducing the S.O.S. response to TcRad51 DNA interaction. Rad51 knockout Saccharomyces cerevisiae was unable to grow in the presence of TcRad51 protein. This phenotype may be caused by the incapacity of this strain to repair double strand breakage, which is masked by TcRad51 protein. In addition to the characterization of the TcRad51 gene, we investigated aspects of molecular evolution in ten strains of T. cruzi. A high degree of conservation was observed in the 5-terminal (359 base pairs fragment) of TcRad51 gene. These strains were divided in three groups according to the phylogenetic analyses based on this fragment. Furthermore, two strains, 167 and 115, may be considered as hybrids because they have alleles that belong to two different groups. The results presented here open new perspectives to the understanding of an important aspect of the T. cruzi biology related to DNA metabolism. | |
