Nuclear DNA Replication in Trypanosomatids: There Are No Easy Methods for Solving Difficult Problems

Silva, Marcelo S. da; Pavani, Raphael Souza; Damasceno, Jeziel D.; Marques, Catarina A.; McCulloch, Richard; Tosi, Luiz Ricardo Orsini; Elias, Maria Carolina

doi:10.1016/j.pt.2017.08.002

Cited by 36 publications

(34 citation statements)

References 96 publications

(168 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition, while SNS-seq has been performed in asynchronous cells, MFA-seq has been used with enriched populations for specific cell cycle populations. Nonetheless, it remains unclear if and how the considerably variant predictions of DNA replication activity by these different methodologies can fit together (discussed at length in recent reviews) (72,74,81). Having said this, it is very possible that the description of subtelomeric DNA replication provides at least a partial explanation for single DNA molecules, visualised by DNA combing, that display two regions of DNA synthesis (18,19).…”

Section: Discussionmentioning

confidence: 99%

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Damasceno

Marques

Beraldi

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

Once every cell cycle, DNA replication takes place to allow cells to duplicate their genome and segregate the two resulting copies into offspring cells. In eukaryotes, the number of DNA replication initiation loci, termed origins, is proportional to chromosome size. However, previous studies have suggested that in Leishmania, a group of single-celled eukaryotic parasites, DNA replication starts from just a single origin per chromosome, which is predicted to be insufficient to secure complete genome duplication within S phase.Here, we show that the paucity of origins activated in early S phase is balanced by DNA synthesis activity outside S phase. Simultaneous recruitment of acetylated histone H3 (AcH3), modified base J and the kinetochore factor KKT1 is exclusively found at the origins used in early S phase, while subtelomeric DNA replication can only be linked to AcH3 and displays persistent activity through the cell cycle, including in G2/M and G1 phases. We also show that subtelomeric DNA replication, unlike replication from the previously mapped origins, is sensitive to hydroxyurea and dependent on subunits of the 9-1-1 complex.Our work indicates that Leishmania genome transmission relies on an unconventional DNA replication programme, which may have implications for genome stability in this important parasite. sequence read depth in the former relative to the latter. As result, DNA synthesis very late in S-phase, or that occurs outside of S-phase, would escape detection. To test this, we modified the MFA-seq approach in order that DNA content enrichment could be calculated in replicating cells relative to naturally occurring non-replicative cells. Mapping origins of replication by calculating DNA enrichment in exponentially growing cells versus cells in a stationary state has been used successfully in bacteria (42) and yeast (43). Thus, we reasoned that L. major in stationary phase could also serve as a non-replicative control for normalization in MFA-seq analysis compared with cells that are growing exponentially. To test this, we used flow cytometry to compare DNA content of exponentially growing and stationary phase cells and, in addition, compared the capacity of the two cell populations to incorporate the thymidine analogue 5-ethynyl-2'deoxyuridine (EdU; Fig. 1A,B). Flow cytometry showed that the proportion of cells in S phase (with DNA content between 1C and 2C) was substantially lower in a population of stationary cells compared with exponentially growing cells (Fig. 1A). Concomitantly, stationary phase cells, unlike exponentially growing cells, failed to incorporate EdU, even upon relatively long periods of incubation ( Fig. 1B). Thus, L. major cells in stationary phase do not perform any detectable DNA synthesis and were therefore deemed suitable to be used as the non-replicative sample in MFA-seq analysis.Next, we compared MFA-seq profiles using read depth ratios from FACS-sorted early S (ES) and G2 cells, and from exponentially growing cells (EXP) and stationary (STA) cells (Fig. 1C). As reported ...

show abstract

Section: Discussionmentioning

confidence: 99%

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Damasceno

Marques

Beraldi

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…TRYPANOSOMATIDS are unicellular eukaryotic parasites considered to be evolutionarily successful, not only for their ability to infect numerous types of organisms but also because they diverged early in the evolutionary scale, approximately 200-500 million years ago (El-Sayed et al 2005;Flegontov et al 2013;da Silva et al 2017;Simpson et al 2004). These organisms are represented by the agent of African sleeping sickness, Trypanosoma brucei, several species of Leishmania spp., which cause distinct types of leishmaniasis, and the etiological agent of Chagas disease, Trypanosoma cruzi.…”

mentioning

confidence: 99%

The Dynamics of Replication in Trypanosoma cruzi Parasites by Single‐Molecule Analysis

Araujo

Calderano

Elias

2018

J Eukaryotic Microbiology

Self Cite

View full text Add to dashboard Cite

Here, we investigated the features of replication in Trypanosoma cruzi epimastigotes based on fork speed progression, which is influenced by distinct features such as DNA polymerase rate, susceptibility to DNA damage and repair, secondary structures, transcription and chromatin state. Although T. cruzi exhibits a mean fork speed (2.05 ± 0.10 kb/min) very similar to other trypanosomatids, we found that the majority of DNA molecules replicated more slowly, with a frequency distribution approximately 1 kb/min. This frequency distribution analysis provides more information about the replication profile of this organism.

show abstract

“…However, none of these studies indicated that replication-transcription conflicts contributed to the firing of origins, which, in model eukaryotes, is an apparently wellregulated event (6). Based on this, we have proposed that DNA damage is generated after replication and transcription find each other in conflicting directions, which signals to initiate the phosphorylation of a nearby histone H2A, either by ATR or ATM kinases (14,34,78). Moreover, the hybrid R-loop structure is likely formed and maintained in the collision region.…”

Section: Discussionmentioning

confidence: 99%

“…However, the same study identified many more ORC binding sites than activated origins (16), which can be easily explained by the fact that MFA-seq (genome-wide analysis) is a technique that provides a result based on population analysis. Thus, the set of activated origins that matches with ORC binding sites can be classified as constitutive, and the remaining ORC binding sites are probably potential sites for the firing of non-constitutive origins (flexible or dormant) (6,14).…”

Section: Edu Allows a More Accurate Estimation Of S Phase Duration Inmentioning

confidence: 99%

“…and Leishmania spp., which are the causative agents of devastating diseases that threaten millions of people around the world (12,13). Trypanosoma brucei, one of the parasites known as African trypanosomes, has the best-studied DNA and RNA synthesis, although this knowledge is limited relative to that of model eukaryotes (14). The duration of the by using a most sensitive thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU), to monitor DNA replication, although there are still no similar assays for T. brucei TREU927 (15).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Transcription activity contributes to the firing of non-constitutive origins in Trypanosoma brucei

Silva¹,

Cayres-Silva²,

Vitarelli³

et al. 2018

Preprint

Self Cite

View full text Add to dashboard Cite

The cosynthesis of DNA and RNA potentially generates conflicts between replication and transcription, which can lead to genomic instability. In trypanosomatids, eukaryotic parasites that perform polycistronic transcription, this phenomenon and its consequences have not yet been investigated. Here, using equations and computational analysis we demonstrated that the number of constitutive origins mapped in the Trypanosoma brucei genome is close to the minimum required to complete replication within S phase duration. However, taking into account the location of these origins in the genome, the replication in due time becomes virtually impossible, making it necessary to activate non-constitutive origins. Moreover, computational and biological assays pointed to transcription being responsible for activating non-constitutive origins. Together, our results suggest that transcription action through conflicts with replication contributes to the firing of non-constitutive origins, maintaining the robustness of S phase duration. The usage of this entire pool of origins seems to be of paramount importance for the survival of this parasite that infects million people around the world since it contributes to the maintenance of the replication of its DNA.

show abstract

Nuclear DNA Replication in Trypanosomatids: There Are No Easy Methods for Solving Difficult Problems

Cited by 36 publications

References 96 publications

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

The Dynamics of Replication in Trypanosoma cruzi Parasites by Single‐Molecule Analysis

Transcription activity contributes to the firing of non-constitutive origins in Trypanosoma brucei

Contact Info

Product

Resources

About