Navigating the boundaries between metabolism and epigenetics in trypanosomes.

Epigenetic marks enable cells to acquire new biological features that favor their adaptation to environmental changes. These marks are chemical modifications on chromatin-associated proteins and nucleic acids that lead to changes in the chromatin landscape and may eventually affect gene expression. The chemical tags of these epigenetic marks are comprised of intermediate cellular metabolites.

Life in plastic, it's fantastic! How exploit genome instability to shape gene expression.

are kinetoplastid pathogens that cause leishmaniasis, a debilitating and potentially life-threatening infection if untreated. Unusually, regulate their gene expression largely post-transcriptionally due to the arrangement of their coding genes into polycistronic transcription units that may contain 100s of functionally unrelated genes. Yet, are capable of rapid and responsive changes in gene expression to challenging environments, often instead correlating with dynamic changes in their genome composition, ranging from chromosome and gene copy number variations to the generation of extrachromosomal DNA and the accumulation of point mutations.

Specific Human ATR and ATM Inhibitors Modulate Single Strand DNA Formation in Exposed to Oxidative Agent.

parasites are the causative agents of a group of neglected tropical diseases known as leishmaniasis. The molecular mechanisms employed by these parasites to adapt to the adverse conditions found in their hosts are not yet completely understood. DNA repair pathways can be used by to enable survival in the interior of macrophages, where the parasite is constantly exposed to oxygen reactive species.

New Insights into the Mechanism of Action of the Cyclopalladated Complex (CP2) in : Calcium Dysregulation, Mitochondrial Dysfunction, and Cell Death.

The current treatment of leishmaniasis is based on a few drugs that present several drawbacks, such as high toxicity, difficult administration route, and low efficacy. These disadvantages raise the necessity to develop novel antileishmanial compounds allied with a comprehensive understanding of their mechanisms of action. Here, we elucidate the probable mechanism of action of the antileishmanial binuclear cyclopalladated complex [Pd(dmba)(μ-N)] (CP2) in Leishmania amazonensis.

DNA Topoisomerase 3α Is Involved in Homologous Recombination Repair and Replication Stress Response in .

DNA topoisomerases are enzymes that modulate DNA topology. Among them, topoisomerase 3α is engaged in genomic maintenance acting in DNA replication termination, sister chromatid separation, and dissolution of recombination intermediates. To evaluate the role of this enzyme in , the etiologic agent of Chagas disease, a topoisomerase 3α knockout parasite (TcTopo3α KO) was generated, and the parasite growth, as well as its response to several DNA damage agents, were evaluated.

Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication.

Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins.

DiCre-Based Inducible Gene Expression.

Induction of gene expression is a valuable approach for functional studies since it allows for the assessment of phenotypes without the need for clonal selection. Inducible expression can find a wide range of applications, from the study of essential genes to the characterization of overexpression of genes of interest. Here, we describe a detailed protocol for the use of the DiCre-based inducible gene expression system in Leishmania parasites.

The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions.

In Trypanosoma cruzi, the etiologic agent of Chagas disease, Rad51 (TcRad51) is a central enzyme for homologous recombination. Here we describe the different roles of TcRad51 in DNA repair. Epimastigotes of T.

Conditional genome engineering reveals canonical and divergent roles for the Hus1 component of the 9-1-1 complex in the maintenance of the plastic genome of Leishmania.

Leishmania species are protozoan parasites whose remarkably plastic genome limits the establishment of effective genetic manipulation and leishmaniasis treatment. The strategies used by Leishmania to maintain its genome while allowing variability are not fully understood. Here, we used DiCre-mediated conditional gene deletion to show that HUS1, a component of the 9-1-1 (RAD9-RAD1-HUS1) complex, is essential and is required for a G2/M checkpoint.

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

In trypanosomatids, etiological agents of devastating diseases, replication is robust and finely controlled to maintain genome stability and function in stressful environments. However, these parasites encode several replication protein components and complexes that show potentially variant composition compared with model eukaryotes. This review focuses on the advances made in recent years regarding the differences and peculiarities of the replication machinery in trypanosomatids, including how such divergence might affect DNA replication dynamics and the replication stress response.

A DiCre recombinase-based system for inducible expression in Leishmania major.

Here we present the establishment of an inducible system based on the dimerizable Cre recombinase (DiCre) for controlled gene expression in the protozoan parasite Leishmania. Rapamycin-induced DiCre activation promoted efficient flipping and expression of gene products in a time and dose-dependent manner. The DiCre flipping activity induced the expression of target genes from both integrated and episomal contexts broadening the applicability of the system.

A transposon-based tool for transformation and mutagenesis in trypanosomatid protozoa.

The ability of transposable elements to mobilize across genomes and affect the expression of genes makes them exceptional tools for genetic manipulation methodologies. Several transposon-based systems have been modified and incorporated into shuttle mutagenesis approaches in a variety of organisms. We have found that the Mos1 element, a DNA transposon from Drosophila mauritiana, is suitable and readily adaptable to a variety of strategies to the study of trypanosomatid parasitic protozoa.

LmHus1 is required for the DNA damage response in Leishmania major and forms a complex with an unusual Rad9 homologue.

Genotoxic stress activates checkpoint-signalling pathways leading to cell cycle arrest and DNA repair. In many eukaryotes, the Rad9-Hus1-Rad1 (9-1-1) checkpoint complex participates in the early steps of the DNA damage response to replicative stress and is a pivotal contributor to genome homeostasis. The remarkable genome plasticity of the protozoan Leishmania hints at a peculiar DNA metabolism in these ancient eukaryotes.

The Hus1 homologue of Leishmania major encodes a nuclear protein that participates in DNA damage response.

The protozoan parasite Leishmania presents a dynamic and plastic genome in which gene amplification and chromosome translocations are common phenomena. Such plasticity hints at the necessity of dependable genome maintenance pathways. Eukaryotic cells have evolved checkpoint control systems that recognize altered DNA structures and halt cell cycle progression allowing DNA repair to take place.

Genome survey sequence analysis and identification of homologs of major surface protease (gp63) genes in Trypanosoma rangeli.

In this study, 222 genome survey sequences were generated for Trypanosoma rangeli strain P07 isolated from an opossum (Didelphis albiventris) in Minas Gerais State, Brazil. T. rangeli sequences were compared by BLASTX (Basic Local Alignment Search Tool X) analysis with the assembled contigs of Leishmania braziliensis, Leishmania infantum, Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi.

A transposon toolkit for gene transfer and mutagenesis in protozoan parasites.

Protozoan parasites affect millions of people around the world. Treatment and control of these diseases are complicated partly due to the intricate biology of these organisms. The interactions of species of Plasmodium, Leishmania and trypanosomes with their hosts are mediated by an unusual control of gene expression that is not fully understood.

Karyotype variability in KP1(+) and KP1(-) strains of Trypanosoma rangeli isolated in Brazil and Colombia.

In the present study, the molecular karyotypes of 12 KP1(+) and KP1(-) Trypanosoma rangeli strains were determined and 10 different molecular markers were hybridized to the chromosomes of the parasite, including seven obtained from T. rangeli [ubiquitin hydrolase (UH), a predicted serine/threonine protein kinase (STK), hexose transporter, hypothetical protein, three anonymous sequences] and three from Trypanosoma cruzi [ubiquitin-conjugating enzyme E2 (UBE2), ribosomal RNA methyltransferase (rRNAmtr), proteasome non-ATPase regulatory subunit 6 (PSMD6)]. Despite intraspecific variation, analysis of the karyotype profiles permitted the division of the T.

Comparative genomic analysis of three Leishmania species that cause diverse human disease.

Leishmania parasites cause a broad spectrum of clinical disease. Here we report the sequencing of the genomes of two species of Leishmania: Leishmania infantum and Leishmania braziliensis. The comparison of these sequences with the published genome of Leishmania major reveals marked conservation of synteny and identifies only approximately 200 genes with a differential distribution between the three species.

Organization of H locus conserved repeats in Leishmania (Viannia) braziliensis correlates with lack of gene amplification and drug resistance.

Resistance to antimonials is a major problem when treating visceral leishmaniasis in India and has already been described for New World parasites. Clinical response to meglumine antimoniate in patients infected with parasites of the Viannia sub-genus can be widely variable, suggesting the presence of mechanisms of drug resistance. In this work, we have compared L.

The use of Tn5 transposable elements in a gene trapping strategy for the protozoan Leishmania.

The use of transposable elements as a gene-trapping strategy is a powerful tool for gene discovery. Herein we describe the development of a transposable system, based on the bacterial Tn5 transposon, which has been used successfully in Leishmania braziliensis. The transposon carries the neomycin phosphotransferase gene, which is expressed only when inserted in-frame with a Leishmania gene present in the target DNA.