In unicellular eukaryotes, such as Saccharomyces cerevisiae, and in multicellular organisms, the replication origin is recognized by the heterohexamer origin recognition complex (ORC) containing six proteins, Orc1 to Orc6, while in members of the domain Archaea, the replication origin is recognized by just one protein, Orc1/Cdc6; the sequence of Orc1/Cdc6 is highly related to those of Orc1 and Cdc6. Similar to Archaea, trypanosomatid genomes contain only one gene encoding a protein named Orc1. Since trypanosome Orc1 is also homologous to Cdc6, in this study we named the Orc1 protein from trypanosomes Orc1/Cdc6. Here we show that the recombinant Orc1/Cdc6 from Trypanosoma cruzi (TcOrc1/Cdc6) and from Trypanosoma brucei (TbOrc1/Cdc6) present ATPase activity, typical of prereplication machinery components. Also, TcOrc1/Cdc6 and TbOrc1/Cdc6 replaced yeast Cdc6 but not Orc1 in a phenotypic complementation assay. The induction of Orc1/Cdc6 silencing by RNA interference in T. brucei resulted in enucleated cells, strongly suggesting the involvement of Orc1/Cdc6 in DNA replication. Orc1/Cdc6 is expressed during the entire cell cycle in the nuclei of trypanosomes, remaining associated with chromatin in all stages of the cell cycle. These results allowed us to conclude that Orc1/Cdc6 is indeed a member of the trypanosome prereplication machinery and point out that trypanosomes carry a prereplication machinery that is less complex than other eukaryotes and closer to archaea.
Approximately ten million people suffer from Chagas disease worldwide, caused by Trypanosoma cruzi, with the disease burden predominately focused in Latin America. Sleeping sickness is another serious health problem, caused by Trypanosoma brucei, especially in sub-Saharan countries. Unfortunately, the drugs currently available to treat these diseases have toxic effects and are not effective against all disease phases or parasite strains. Therefore, there is a clear need for the development of novel drugs and drug targets to treat these diseases. We propose the trypanosome prereplication machinery component, Orc1/Cdc6, as a potential target for drug development. In trypanosomes, Orc1/Cdc6 is involved in nuclear DNA replication, and, despite its involvement in such a conserved process, Orc1/Cdc6 is distinct from mammalian Orc1 and Cdc6 proteins. Moreover, RNAi-mediated silencing of trypanosome Orc1/Cdc6 expression in T. brucei decreased cell survival, indicating that Orc1/Cdc6 is critical for trypanosome survival.
Em eucariotos unicelulares como Saccharomycea cerevisiae, e em organismos multicelulares, a origem de replicação é reconhecida pelo complexo de pré-replicação formado por um complexo denominado ORC (Origin Recognition Complex), as proteínas Cdc6 e Cdt1 e o heterohexâmero MCM (MiniChromosome Mainteinance). Já em organismos do domínio Archaea, a origem de replicação é reconhecida por uma proteína Orc1/Cdc6, que apresenta similaridade com Orc1 e Cdc6, e pelo homohexâmero MCM. De forma semelhante à Archaea, os genomas dos tripanossomatídeos contêm somente um gene que codifica para uma proteína anotada como Orc1. Dada a similaridade entre Orc1 e Cdc6, esta proteína foi por nós denominada Orc1/Cdc6. Nesta tese serão descritos os estudos realizados sobre o componente Orc1/Cdc6 da maquinaria de préreplicação do Trypanosoma cruzi e do Trypanosoma brucei. As proteínas recombinantes de T.cruzi (TcOrc1/Cdc6) e de T.brucei (TbOrc1/Cdc6) foram expressas e ambas proteínas apresentam atividade de ATPase, típica da maquinaria de pré-replicação. As proteínas recombinantes de T.brucei e T.cruzi são capazes de substituir Cdc6, mas não Orc1 em ensaio de complementação em leveduras. A indução do silenciamento do gene de Orc1/Cdc6 por RNAi resulta em células anucleadas com quantidade de DNA equivalente ao estado G1 do ciclo celular, o que sugere fortemente o envolvimento de Orc1/Cdc6 na replicação. Estes resultados sugerem que a maquinaria de pré-replicação dos tripanossomas é diferente daquela encontrada nos demais eucariotos e mais semelhante à de Archaeas. Orc1/Cdc6 é expressa durante todo o ciclo celular das formas replicativas dos tripanossomas, permanecendo associada à cromatina em todas as fases do ciclo celular. No caso do T.cruzi, a proteína Orc1/Cdc6 é expressa não só nas formas replicativas, amastigotas e epimastigotas, mas também nas formas não replicativas tripomastigotas e tripomastigotas metacíclicos. Nas formas não replicativas, a proteína expressa interage fracamente com o DNA. Como o primeiro passo do processo de replicação é a interação da proteína Orc1/Cdc6 com DNA, este resultado sugere que a ausência da interação Orc1/Cdc6-DNA deve contribuir para ausência da duplicação do DNA nas formas infectivas do T. cruzi.
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