Chromosome Localization Changes in the Trypanosoma cruzi Nucleus

Abstract

Chromosome localization in the interphase nuclei of eukaryotes depends on gene replication and transcription. Little is known about chromosome localization in protozoan parasites such as trypanosomes, which have unique mechanisms for the control of gene expression, with most genes being posttranscriptionally regulated. In the present study, we examined where the chromosomes are replicated in Trypanosoma cruzi, the agent of Chagas' disease. The replication sites, identified by the incorporation of 5-bromodeoxyuridine, are located at the nuclear periphery in proliferating epimastigote forms in the early S phase of the cell cycle. When the S phase ends and cells progress through the cell cycle, 5-bromodeoxyuridine labeling is observed in the nuclear interior, suggesting that chromosomes move. We next monitored chromosome locations in different stages of the cell cycle by using a satellite DNA sequence as a probe in a fluorescence in situ hybridization assay. We found two distinct labeling patterns according to the cell cycle stage. The first one is seen in the G 1 phase, in hydroxyurea-arrested epimastigotes or in trypomastigotes, which are differentiated nondividing forms. In all of these forms the satellite DNA is found in dots randomly dispersed in the nucleus. The other pattern is found in cells from the S phase to the G 2 phase. In these cells, the satellite DNA is found preferentially at the nuclear periphery. The labeling at the nuclear periphery disappears only after mitosis. Also, DNA detected with terminal deoxynucleotidyl transferase is found distributed throughout the nuclear space in the G 1 phase but concentrated at the nuclear periphery in the S phase to the G 2 phase. These results strongly suggest that T. cruzi chromosomes move and, after entering the S phase, become constrained at the nuclear periphery, where replication occurs.The eukaryotic nucleus has functional compartments for DNA replication and transcription and for RNA processing (6, 24, 36). Nuclear integrity is important for DNA synthesis initiation and for replication fork formation (7), and disruption of nuclear lamina organization prevents the elongation phase of DNA replication (29). Chromosome localization in the mammalian cell nucleus is also determined by the transcriptional status of genes. Actively transcribed genes are located in the nuclear interior, replicating early in the S phase of the cell cycle, while repressed genes, which correspond to heterochromatic regions, replicate later and are located at the nuclear periphery and around the nucleolus (11,28,32).Visualization of replication sites by labeling of cells with 5-bromodeoxyuridine (BrdU) or biotinylated dUTP (31) during the cell cycle revealed immobile foci probably linked to a nuclear matrix. Over the last 10 to 12 years, detailed information on the organization and dynamics of interphase chromosomes has emerged; most of them have been found associated with repeated domains, such as telomeres and centromeres (10), and related to the cell cycle (16,26). More recently, ...

Unveiling the effects of berenil, a DNA-binding drug, on Trypanosoma cruzi: implications for kDNA ultrastructure and replication

“…To better understand the ultrastructural modifications after treatment with berenil, we used the TdT technique, which specifically labels DNA and was previously employed to study the T. cruzi cell cycle (Elias et al 2002). Our results showed that the kinetoplast electron-lucent areas observed in treated parasites were not labeled, thus indicating that this region did not contain decatenated circles, as initially thought.…”

Trypanosoma cruziDNA replication includes the sequential recruitment of pre-replication and replication machineries close to nuclear periphery

“…We, along with others, have already shown that it is possible to synchronize T. cruzi epimastigote cells with hydroxyurea. 9,17,18 Hydroxyurea inhibits the nucleotide reductase enzyme, thereby consuming dNTPs. In the absence of dNTPs, DNA elongation is blocked and most of the cells are arrested very early in S phase.…”

“…We have also shown that 6 h after hydroxyurea release, cells are in the S phase, replicating their DNA. 9 Analysis of cells at 0 or 6 h after release (S phase) showed that most of these cells display the peripheral pattern for both TcOrc1/Cdc6 and TcPCNA (table 1). These numbers strongly suggest that the peripheral patterns correspond to cells whose DNA is being replicated.…”

“…We have previously shown that the shorter pulse to visualize deoxynucleotide incorporation in T. cruzi epimastigote cells is 2 hours. 9 Therefore, we incubated cells for 2 h with EdU and these cells were analyzed concerning TcPCNA localization. We found that in EdU nonlabeled cells TcPCNA is dispersed through the nuclear space ( fig.…”

“…At the nuclear periphery, chromosomes are concentrated during S phase of the cell cycle. 9 To better understand the organization of DNA replication in this organism, we have examined whether the pre-replication and replication machineries remain constrained at the nuclear periphery during the entire cell cycle or if these replication molecules are recruited to the replication sites in a spatial-temporal manner. As a marker for the pre-replication machinery, we used a molecule named Orc1/Cdc6, which, in trypanosomes, replaces the six Orcs and the Cdc6 normally found in other eukaryotes.…”