RESUMEN
Fluorescent in situ hybridization coupled with immunofluorescence (FISH/IF) is an assay that has been widely used to study DNA-protein interactions. The technique is based on the use of a fluorescent nucleic acid probe and fluorescent antibodies to reveal the localization of a DNA sequence and a specific protein in the cell. The interaction can be inferred by the quantification of the co-localization between the protein and the DNA. Here, we describe a detailed FISH/IF methodology that our group used to study RPA-telomere interaction in the pathogenic protozoa parasite Trypanosoma cruzi.
Asunto(s)
Proteína de Replicación A/metabolismo , Telómero/metabolismo , Trypanosoma cruzi/metabolismo , Técnica del Anticuerpo Fluorescente , Hibridación Fluorescente in Situ , Sondas de Ácido Nucleico/química , Proteínas Protozoarias/metabolismo , Telómero/química , Trypanosoma cruzi/genéticaRESUMEN
Replication protein A (RPA), a heterotrimeric complex, is the major single-stranded DNA binding protein in eukaryotes. Recently, we characterized RPA from Trypanosoma cruzi, showing that it is involved in DNA replication and DNA damage response in this organism. Better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms was observed in TcRPA-2 subunit heterozygous knockout cells, suggesting that RPA is involved in this process. Here, we show that RPA cellular localization changes during the T. cruzi life cycle, with RPA being detected only in the cytoplasm of the metacyclic and bloodstream trypomastigotes. We also identify a nuclear export signal (NES) in the trypanosomatid RPA-2 subunit. Mutations in the negatively charged residues of RPA-2 NES impair the differentiation process, suggesting that RPA exportation affects parasite differentiation into infective forms.
Asunto(s)
Núcleo Celular/metabolismo , Estadios del Ciclo de Vida , Morfogénesis , Proteína de Replicación A/metabolismo , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Enfermedad de Chagas/sangre , Enfermedad de Chagas/parasitología , Simulación por Computador , Citoplasma/metabolismo , Morfogénesis/genética , Señales de Exportación Nuclear/genética , Señales de Exportación Nuclear/fisiología , Proteína de Replicación A/genética , Trypanosoma cruzi/citologíaRESUMEN
In trypanosomatids, etiological agents of devastating diseases, replication is robust and finely controlled to maintain genome stability and function in stressful environments. However, these parasites encode several replication protein components and complexes that show potentially variant composition compared with model eukaryotes. This review focuses on the advances made in recent years regarding the differences and peculiarities of the replication machinery in trypanosomatids, including how such divergence might affect DNA replication dynamics and the replication stress response. Comparing the DNA replication machinery and processes of parasites and their hosts may provide a foundation for the identification of targets that can be used in the development of chemotherapies to assist in the eradication of diseases caused by these pathogens.