RESUMEN
DNA polymerase theta (Polθ), a member of the DNA polymerase family A, exhibits a polymerase C-terminal domain, a central domain, and an N-terminal helicase domain. Polθ plays important roles in DNA repair via its polymerase domain, regulating genome integrity. In addition, in mammals, Polθ modulates origin firing timing and MCM helicase recruitment to chromatin. In contrast, as a model eukaryote, Trypanosoma cruzi exhibits two individual putative orthologs of Polθ in different genomic loci; one ortholog is homologous to the Polθ C-terminal polymerase domain, and the other is homologous to the Polθ helicase domain, called Polθ-polymerase and Polθ-helicase, respectively. A pull-down assay using the T. cruzi component of the prereplication complex Orc1/Cdc6 as bait captured Polθ-helicase from the nuclear extract. Orc1/Cdc6 and Polθ-helicase directly interacted, and Polθ-helicase presented DNA unwinding and ATPase activities. A T. cruzi strain overexpressing the Polθ-helicase domain exhibited a significantly decreased amount of DNA-bound MCM7 and impaired replication origin firing. Taken together, these data suggest that Polθ-helicase modulates DNA replication by directly interacting with Orc1/Cdc6, which reduces the binding of MCM7 to DNA and thereby impairs the firing of replication origins.
Asunto(s)
Cromatina/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Complejo de Reconocimiento del Origen/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/crecimiento & desarrollo , Cromatina/genética , ADN Helicasas/genética , ADN Polimerasa Dirigida por ADN/genética , Humanos , Complejo de Reconocimiento del Origen/genética , Proteínas Protozoarias/genética , Origen de Réplica , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , ADN Polimerasa thetaRESUMEN
Eukaryotic genome duplication relies on origins of replication, distributed over multiple chromosomes, to initiate DNA replication. A recent genome-wide analysis of Trypanosoma brucei, the etiological agent of sleeping sickness, localized its replication origins to the boundaries of multigenic transcription units. To better understand genomic replication in this organism, we examined replication by single molecule analysis of replicated DNA. We determined the average speed of replication forks of procyclic and bloodstream form cells and we found that T. brucei DNA replication rate is similar to rates seen in other eukaryotes. We also analyzed the replication dynamics of a central region of chromosome 1 in procyclic forms. We present evidence for replication terminating within the central part of the chromosome and thus emanating from both sides, suggesting a previously unmapped origin toward the 5' extremity of chromosome 1. Also, termination is not at a fixed location in chromosome 1, but is rather variable. Importantly, we found a replication origin located near an ORC1/CDC6 binding site that is detected after replicative stress induced by hydroxyurea treatment, suggesting it may be a dormant origin activated in response to replicative stress. Collectively, our findings support the existence of more replication origins in T. brucei than previously appreciated.
Asunto(s)
Replicación del ADN/genética , ADN Protozoario/genética , Biología Molecular/métodos , Origen de Réplica/genética , Trypanosoma brucei brucei/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromosomas/genética , Replicación del ADN/efectos de los fármacos , Citometría de Flujo , Genoma de Protozoos/genética , Hidroxiurea/farmacología , Cinética , Estadios del Ciclo de Vida/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Complejo de Reconocimiento del Origen/genética , Complejo de Reconocimiento del Origen/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Factores de Tiempo , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
Trypanosoma cruzi alternates between replicative and non-replicative stages. We analyzed the expression of components of the pre-replication machinery TcORC1/CDC6 and TcMCM7 and their interaction with DNA in all T. cruzi stages. TcORC1/CDC6 remains in the nuclear space during all stages of the life cycle and interacts with DNA in the replicative stages; however, it does not bind to DNA in the non-replicative forms. Moreover, TcMCM7 is not present in the non-replicative stages. These data suggest that the lacking of DNA replication during the T. cruzi life cycle may be a consequence of the blocking of TcORC1/CDC6-DNA interaction and of the down regulation of the TcMCM7 expression.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Complejo de Reconocimiento del Origen/metabolismo , Trypanosoma cruzi/crecimiento & desarrollo , Animales , Proteínas de Ciclo Celular/genética , Replicación del ADN , Estadios del Ciclo de Vida , Componente 7 del Complejo de Mantenimiento de Minicromosoma/genética , Complejo de Reconocimiento del Origen/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismoRESUMEN
In eukaryotes, many nuclear processes are spatially compartmentalized. Previously, we have shown that in Trypanosoma cruzi, an early-divergent eukaryote, DNA replication occurs at the nuclear periphery where chromosomes remain constrained during the S phase of the cell cycle. We followed Orc1/Cdc6, a pre-replication machinery component and the proliferating cell nuclear antigen (PCNA), a component of replication machinery, during the cell cycle of this protozoon. We found that, at the G(1) stage, TcOrc1/Cdc6 and TcPCNA are dispersed throughout the nuclear space. During the G(1)/S transition, TcOrc1/Cdc6 migrates to a region close to nuclear periphery. At the onset of S phase, TcPCNA is loaded onto the DNA and remains constrained close to nuclear periphery. Finally, in G(2), mitosis and cytokinesis, TcOrc1/Cdc6 and TcPCNA are dispersed throughout the nuclear space. Based on these findings, we propose that DNA replication in T. cruzi is accomplished by the organization of functional machineries in a spatial-temporal manner.
Asunto(s)
Núcleo Celular/metabolismo , Replicación del ADN , ADN Protozoario/biosíntesis , Trypanosoma cruzi/citología , Trypanosoma cruzi/metabolismo , Transporte Activo de Núcleo Celular , Proteínas de Ciclo Celular/metabolismo , ADN Protozoario/metabolismo , Fase G1 , Complejo de Reconocimiento del Origen/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Protozoarias/metabolismo , Fase SRESUMEN
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.