<i>Trypanosoma cruzi</i>DNA replication includes the sequential recruitment of pre-replication and replication machineries close to nuclear periphery

Calderano, Simone Guedes; Godoy, Patricia Diogo de Melo; Motta, Maria Cristina M.; Mortara, Renato Arruda; Schenkman, Sérgio; Elias, Maria Carolina

doi:10.4161/nucl.2.2.15134

Cited by 19 publications

(22 citation statements)

References 29 publications

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“…proteins that act in DNA replication), it is necessary to reduce misinterpretation that can generate a bias in the equations that measure the percentage of co‐localization (Calderano et al. ; Manders et al. ).…”

Section: Discussionmentioning

confidence: 99%

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Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S, and G2 Phases of the Cell Cycle in Trypanosomatids

Silva

Muñoz

Armelin

et al. 2017

J Eukaryotic Microbiology

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Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU- and EdU-incorporated parasites with the respective standard detection approaches: indirect immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and "click" chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re-estimated the phases G1, S, G2 + M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring.

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Section: Discussionmentioning

confidence: 99%

“…However, due to their time‐consuming detection and the need for genomic DNA denaturation to provide access for anti‐BrdU antibodies, current studies are commonly being performed using the other thymidine analog, 5‐ethynyl‐2′‐deoxyuridine (EdU) (Calderano et al. ; Salic and Mitchison ; da Silva et al. ).…”

mentioning

confidence: 99%

Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S, and G2 Phases of the Cell Cycle in Trypanosomatids

Silva

Muñoz

Armelin

et al. 2017

J Eukaryotic Microbiology

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“…Amastigotes proliferate inside the infected cells until they transform into non-replicative trypomastigotes. The life cycle is completed when an insect vector bites an infected mammalian host and takes up trypomastigotes within the blood that then transform into epimastigotes inside the insect gut ([2]). Although it has been previously described that some stressors, such as acidic pH and starvation, trigger the transition from one form to another [3], the molecular bases involved in this response remain to be elucidated, such as which molecules are sensors or transducers of these differentiation pathways.…”

Section: Introductionmentioning

confidence: 99%

Replication Protein A Presents Canonical Functions and Is Also Involved in the Differentiation Capacity of Trypanosoma cruzi

et al. 2016

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Replication Protein A (RPA), the major single stranded DNA binding protein in eukaryotes, is composed of three subunits and is a fundamental player in DNA metabolism, participating in replication, transcription, repair, and the DNA damage response. In human pathogenic trypanosomatids, only limited studies have been performed on RPA-1 from Leishmania. Here, we performed in silico, in vitro and in vivo analysis of Trypanosoma cruzi RPA-1 and RPA-2 subunits. Although computational analysis suggests similarities in DNA binding and Ob-fold structures of RPA from T. cruzi compared with mammalian and fungi RPA, the predicted tridimensional structures of T. cruzi RPA-1 and RPA-2 indicated that these molecules present a more flexible tertiary structure, suggesting that T. cruzi RPA could be involved in additional responses. Here, we demonstrate experimentally that the T. cruzi RPA complex interacts with DNA via RPA-1 and is directly related to canonical functions, such as DNA replication and DNA damage response. Accordingly, a reduction of TcRPA-2 expression by generating heterozygous knockout cells impaired cell growth, slowing down S-phase progression. Moreover, heterozygous knockout cells presented a better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms and metacyclic trypomastigote infection. Taken together, these findings indicate the involvement of TcRPA in the metacyclogenesis process and suggest that a delay in cell cycle progression could be linked with differentiation in T. cruzi.

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“…In the S phase, it is localized in subnuclear foci at the nucleus periphery, while in G 2 /M, it is more widely distributed. This feature, which is conserved also in higher eukaryotes, indicates the presence of replication factories in specific sub-locations in Leishmania and related species (Calderano et al, 2011;Kumar et al, 2009;Sch€ onenberger et al, 2015). Also two PI3KC paralogues are present in Leishmania spp (Table 3), but little is known about their in vivo roles.…”

Section: Elongationmentioning

confidence: 99%

“…PCNA has been detected throughout the cell cycle in L. donovani, T. brucei and T. cruzi (Calderano et al, 2011;Kaufmann et al, 2012;Kumar et al, 2009;Valenciano et al, 2015) and its subnuclear expression pattern varies during the cell cycle. In the S phase, it is localized in subnuclear foci at the nucleus periphery, while in G 2 /M, it is more widely distributed.…”

Section: Elongationmentioning

confidence: 99%

Nuclear DNA replication and repair in parasites of the genusLeishmania: Exploiting differences to develop innovative therapeutic approaches

Uzcanga

Lara

Gutiérrez

et al. 2016

Critical Reviews in Microbiology

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

Cited by 19 publications

References 29 publications

Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S, and G2 Phases of the Cell Cycle in Trypanosomatids

Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S, and G2 Phases of the Cell Cycle in Trypanosomatids

Replication Protein A Presents Canonical Functions and Is Also Involved in the Differentiation Capacity of Trypanosoma cruzi

Nuclear DNA replication and repair in parasites of the genusLeishmania: Exploiting differences to develop innovative therapeutic approaches

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