Determination of the Ribonucleotide Content of mtDNA Using Alkaline Gels.

Impaired mitochondrial DNA (mtDNA) maintenance, due to, e.g., defects in the replication machinery or an insufficient dNTP supply, underlies a number of mitochondrial disorders. The normal process of mtDNA replication leads to the incorporation of multiple single ribonucleotides (rNMPs) per mtDNA molecule. Given that embedded rNMPs alter the stability and properties of the DNA, they may have consequences for mtDNA maintenance and thereby for mitochondrial disease. They also serve as a readout of the intramitochondrial NTP/dNTP ratios. In this chapter, we describe a method for the determination of mtDNA rNMP content using alkaline gel electrophoresis and Southern blotting. This procedure is suited for the analysis of mtDNA in total genomic DNA preparations as well as in purified form. Moreover, it can be performed using equipment found in most biomedical laboratories, allows the simultaneous analysis of 10-20 samples depending on the gel system employed, and can be modified for the analysis of other mtDNA modifications.

Mitochondrial DNA Instability in Mammalian Cells.

Significance: The small, multicopy mitochondrial genome (mitochondrial DNA [mtDNA]) is essential for efficient energy production, as alterations in its coding information or a decrease in its copy number disrupt mitochondrial ATP synthesis. However, the mitochondrial replication machinery encounters numerous challenges that may limit its ability to duplicate this important genome and that jeopardize mtDNA stability, including various lesions in the DNA template, topological stress, and an insufficient nucleotide supply. Recent Advances: An ever-growing array of DNA repair or maintenance factors are being reported to localize to the mitochondria. We review current knowledge regarding the mitochondrial factors that may contribute to the tolerance or repair of various types of changes in the mitochondrial genome, such as base damage, incorporated ribonucleotides, and strand breaks. We also discuss the newly discovered link between mtDNA instability and activation of the innate immune response. Critical Issues: By which mechanisms do mitochondria respond to challenges that threaten mtDNA maintenance? What types of mtDNA damage are repaired, and when are the affected molecules degraded instead? And, finally, which forms of mtDNA instability trigger an immune response, and how? Future Directions: Further work is required to understand the contribution of the DNA repair and damage-tolerance factors present in the mitochondrial compartment, as well as the balance between mtDNA repair and degradation. Finally, efforts to understand the events underlying mtDNA release into the cytosol are warranted. Pursuing these and many related avenues can improve our understanding of what goes wrong in mitochondrial disease. Antioxid. Redox Signal. 36, 885-905.

The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method.

The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR, but is typically assessed under conditions that fail to provide information on the individual mtDNA strands. Using denaturing gel electrophoresis, we show that commonly-used isolation procedures generate mtDNA containing several single-strand breaks per strand. Through systematic comparison of DNA isolation methods, we identify a procedure yielding the highest integrity of mtDNA that we demonstrate displays improved performance in downstream assays. Our results highlight the importance of isolation method choice, and serve as a resource to researchers requiring high-quality mtDNA from solid tissues.

Importance of Angomonas deanei KAP4 for kDNA arrangement, cell division and maintenance of the host-bacterium relationship.

Angomonas deanei coevolves in a mutualistic relationship with a symbiotic bacterium that divides in synchronicity with other host cell structures. Trypanosomatid mitochondrial DNA is contained in the kinetoplast and is composed of thousands of interlocked DNA circles (kDNA). The arrangement of kDNA is related to the presence of histone-like proteins, known as KAPs (kinetoplast-associated proteins), that neutralize the negatively charged kDNA, thereby affecting the activity of mitochondrial enzymes involved in replication, transcription and repair. In this study, CRISPR-Cas9 was used to delete both alleles of the A. deanei KAP4 gene. Gene-deficient mutants exhibited high compaction of the kDNA network and displayed atypical phenotypes, such as the appearance of a filamentous symbionts, cells containing two nuclei and one kinetoplast, and division blocks. Treatment with cisplatin and UV showed that Δkap4 null mutants were not more sensitive to DNA damage and repair than wild-type cells. Notably, lesions caused by these genotoxic agents in the mitochondrial DNA could be repaired, suggesting that the kDNA in the kinetoplast of trypanosomatids has unique repair mechanisms. Taken together, our data indicate that although KAP4 is not an essential protein, it plays important roles in kDNA arrangement and replication, as well as in the maintenance of symbiosis.

DNA lesions and repair in trypanosomatids infection.

Pathological processes such as bacterial, viral and parasitic infections can generate a plethora of responses such as, but not restricted to, oxidative stress that can be harmful to the host and the pathogen. This stress occurs when there is an imbalance between reactive oxygen species produced and antioxidant factors produced in response to the infection. This imbalance can lead to DNA lesions in both infected cells as well as in the pathogen. The effects of the host response on the parasite lead to several kinds of DNA damage, causing alterations in the parasite's metabolism; the reaction and sensitivity of the parasite to these responses are related to the DNA metabolism and life cycle of each parasite. The present review will discuss the survival strategies developed by host cells and Trypanosoma cruzi, focusing on the DNA repair mechanisms of these organisms throughout infection including the relationship between DNA damage, stress response features, and the unique characteristics of these diseases.

The Influence of Recombinational Processes to Induce Dormancy in Trypanosoma cruzi.

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that affects around 8 million people worldwide. Chagas disease can be divided into two stages: an acute stage with high parasitemia followed by a low parasitemia chronic stage. Recently, the importance of dormancy concerning drug resistance in T. cruzi amastigotes has been shown. Here, we quantify the percentage of dormant parasites from different T. cruzi DTUs during their replicative epimastigote and amastigote stages. For this study, cells of T. cruzi CL Brener (DTU TcVI); Bug (DTU TcV); Y (DTU TcII); and Dm28c (DTU TcI) were used. In order to determine the proliferation rate and percentage of dormancy in epimastigotes, fluorescent-labeled cells were collected every 24 h for flow cytometer analysis, and cells showing maximum fluorescence after 144 h of growth were considered dormant. For the quantification of dormant amastigotes, fluorescent-labeled trypomastigotes were used for infection of LLC-MK2 cells. The number of amastigotes per infected LLC-MK2 cell was determined, and those parasites that presented fluorescent staining after 96 h of infection were considered dormant. A higher number of dormant cells was observed in hybrid strains when compared to non-hybrid strains for both epimastigote and amastigote forms. In order to investigate, the involvement of homologous recombination in the determination of dormancy in T. cruzi, we treated CL Brener cells with gamma radiation, which generates DNA lesions repaired by this process. Interestingly, the dormancy percentage was increased in gamma-irradiated cells. Since, we have previously shown that naturally-occurring hybrid T. cruzi strains present higher transcription of RAD51-a key gene in recombination process -we also measured the percentage of dormant cells from T. cruzi clone CL Brener harboring single knockout for RAD51. Our results showed a significative reduction of dormant cells in this T. cruzi CL Brener RAD51 mutant, evidencing a role of homologous recombination in the process of dormancy in this parasite. Altogether, our data suggest the existence of an adaptive difference between T. cruzi strains to generate dormant cells, and that homologous recombination may be important for dormancy in this parasite.

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. cruzi overexpressing TcRAD51 presented abundant TcRad51-labeled foci before gamma irradiation treatment, and a faster growth recovery when compared to single-knockout epimastigotes for RAD51. Overexpression of RAD51 also promoted increased resistance against hydrogen peroxide treatment, while the single-knockout epimastigotes for RAD51 exhibited increased sensitivity to this oxidant agent, which indicates a role for this gene in the repair of DNA oxidative lesions. In contrast, TcRad51 was not involved in the repair of crosslink lesions promoted by UV light and cisplatin treatment. Also, RAD51 single-knockout epimastigotes showed a similar growth rate to that exhibited by wild-type ones after treatment with hydroxyurea, but an increased sensitivity to methyl methane sulfonate. Besides its role in epimastigotes, TcRad51 is also important during mammalian infection, as shown by increased detection of T. cruzi cells overexpressing RAD51, and decreased detection of single-knockout cells for RAD51, in both fibroblasts and macrophages infected with amastigotes. Besides that, RAD51-overexpressing parasites infecting mice also presented increased infectivity and higher resistance against benznidazole. We thus show that TcRad51 is involved in the repair of DNA double strands breaks and oxidative lesions in two different T. cruzi developmental stages, possibly playing an important role in the infectivity of this parasite.

The recombinase Rad51 plays a key role in events of genetic exchange in Trypanosoma cruzi.

Detection of genetic exchange has been a limiting factor to deepen the knowledge on the mechanisms by which Trypanosoma cruzi is able to generate progeny and genetic diversity. Here we show that incorporation of halogenated thymidine analogues, followed by immunostaining, is a reliable method not only to detect T. cruzi fused-cell hybrids, but also to quantify their percentage in populations of this parasite. Through this approach, we were able to detect and quantify fused-cell hybrids of T. cruzi clones CL Brener and Y. Given the increased detection of fused-cell hybrids in naturally-occurring hybrid CL Brener strain, which displays increased levels of RAD51 and BRCA2 transcripts, we further investigated the role of Rad51 - a recombinase involved in homologous recombination - in the process of genetic exchange. We also verified that the detection of fused-cell hybrids in T. cruzi overexpressing RAD51 is increased when compared to wild-type cells, suggesting a key role for Rad51 either in the formation or in the stabilization of fused-cell hybrids in this organism.

Assessment of genetic mutation frequency induced by oxidative stress in Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas disease, a public health challenge due to its morbidity and mortality rates, which affects around 6-7 million people worldwide. Symptoms, response to chemotherapy, and the course of Chagas disease are greatly influenced by T. cruzi's intra-specific variability. Thus, DNA mutations in this parasite possibly play a key role in the wide range of clinical manifestations and in drug sensitivity. Indeed, the environmental conditions of oxidative stress faced by T. cruzi during its life cycle can generate genetic mutations. However, the lack of an established experimental design to assess mutation rates in T. cruzi precludes the study of conditions and mechanisms that potentially produce genomic variability in this parasite. We developed an assay that employs a reporter gene that, once mutated in specific positions, convert G418-sensitive into G418-insenstitive T. cruzi. We were able to determine the frequency of DNA mutations in T. cruzi exposed and non-exposed to oxidative insults assessing the number of colony-forming units in solid selective media after plating a defined number of cells. We verified that T. cruzi's spontaneous mutation frequency was comparable to those found in other eukaryotes, and that exposure to hydrogen peroxide promoted a two-fold increase in T. cruzi's mutation frequency. We hypothesize that genetic mutations in T. cruzi can arise from oxidative insults faced by this parasite during its life cycle.

Analysis of DNA Exchange Using Thymidine Analogs (ADExTA) in Trypanosoma cruzi.

Trypanosoma cruzi is a protozoan parasite belonging to the Trypanosomatidae family. Although the trypanosomatids multiply predominantly by clonal generation, the presence of DNA exchange in some of them has been puzzling researchers over the years, mainly because it may represent a novel form that these organisms use to gain variability. Analysis of DNA Exchange using Thymidine Analogs (ADExTA) is a method that allows the in vitro detection and measurement of rates of DNA exchange, particularly in trypanosomatid cells, in a rapid and simple manner by indirect immunofluorescence assay (IFA). The method can be used to detect DNA exchange within one trypanosomatid lineage or among different lineages by paired analysis. The principle of this assay is based on the incorporation of two distinguishable halogenated thymidine analogs called 5'-chloro-2'-deoxyuridine (CldU) and 5'-iodo-2'-deoxyuridine (IdU) during DNA replication. After mixing the two cell cultures that had been previously incorporated with CldU and IdU separately, the presence of these unusual deoxynucleosides in the genome can be detected by specific antibodies. For this, a DNA denaturation step is required to expose the sites of thymidine analogs incorporated. Subsequently, a secondary reaction using fluorochrome-labeled antibodies will generate distinct signals under fluorescence analysis. By using this method, DNA exchange verification (i.e., the presence of both CldU and IdU in the same cell) is possible using a standard fluorescence microscope. It typically takes 2-3 days from the thymidine analogs incorporation to results. Of note, ADExTA is relatively cheap and does not require transfections or harsh genetic manipulation. These features represent an advantage when compared to other time-consuming protocols that demand DNA manipulation to introduce distinct drug-resistance markers in different cells for posterior selection.

Analysis of DNA Exchange Using Thymidine Analogs (ADExTA) in Trypanosoma cruzi

Trypanosoma cruzi is a protozoan parasite belonging to the Trypanosomatidae family. Although the trypanosomatids multiply predominantly by clonal generation, the presence of DNA exchange in some of them has been puzzling researchers over the years, mainly because it may represent a novel form that these organisms use to gain variability. Analysis of DNA Exchange using Thymidine Analogs (ADExTA) is a method that allows the in vitro detection and measurement of rates of DNA exchange, particularly in trypanosomatid cells, in a rapid and simple manner by indirect immunofluorescence assay (IFA). The method can be used to detect DNA exchange within one trypanosomatid lineage or among different lineages by paired analysis. The principle of this assay is based on the incorporation of two distinguishable halogenated thymidine analogs called 5'-chloro-2'-deoxyuridine (CldU) and 5'-iodo-2'-deoxyuridine (IdU) during DNA replication. After mixing the two cell cultures that had been previously incorporated with CldU and IdU separately, the presence of these unusual deoxynucleosides in the genome can be detected by specific antibodies. For this, a DNA denaturation step is required to expose the sites of thymidine analogs incorporated. Subsequently, a secondary reaction using fluorochrome-labeled antibodies will generate distinct signals under fluorescence analysis. By using this method, DNA exchange verification (i.e., the presence of both CldU and IdU in the same cell) is possible using a standard fluorescence microscope. It typically takes 2-3 days from the thymidine analogs incorporation to results. Of note, ADExTA is relatively cheap and does not require transfections or harsh genetic manipulation. These features represent an advantage when compared to other time-consuming protocols that demand DNA manipulation to introduce distinct drug-resistance markers in different cells for posterior selection.

The recombinase Rad51 plays a key role in events of genetic exchange in Trypanosoma cruzi

Detection of genetic exchange has been a limiting factor to deepen the knowledge on the mechanisms by which Trypanosoma cruzi is able to generate progeny and genetic diversity. Here we show that incorporation of halogenated thymidine analogues, followed by immunostaining, is a reliable method not only to detect T. cruzi fused-cell hybrids, but also to quantify their percentage in populations of this parasite. Through this approach, we were able to detect and quantify fused-cell hybrids of T. cruzi clones CL Brener and Y. Given the increased detection of fused-cell hybrids in naturally-occurring hybrid CL Brener strain, which displays increased levels of RAD51 and BRCA2 transcripts, we further investigated the role of Rad51 – a recombinase involved in homologous recombination – in the process of genetic exchange. We also verified that the detection of fused-cell hybrids in T. cruzi overexpressing RAD51 is increased when compared to wild-type cells, suggesting a key role for Rad51 either in the formation or in the stabilization of fused-cell hybrids in this organism.

Analysis of DNA exchange using Thymidine analogs (ADExTA) in Trypanosoma cruzi

Trypanosoma cruzi is a protozoan parasite belonging to the Trypanosomatidae family. Although the trypanosomatids multiply predominantly by clonal generation, the presence of DNA exchange in some of them has been puzzling researchers over the years, mainly because it may represent a novel form that these organisms use to gain variability. Analysis of DNA Exchange using Thymidine Analogs (ADExTA) is a method that allows the in vitro detection and measurement of rates of DNA exchange, particularly in trypanosomatid cells, in a rapid and simple manner by indirect immunofluorescence assay (IFA). The method can be used to detect DNA exchange within one trypanosomatid lineage or among different lineages by paired analysis. The principle of this assay is based on the incorporation of two distinguishable halogenated thymidine analogs called 5'-chloro-2'-deoxyuridine (CldU) and 5'-iodo-2'-deoxyuridine (IdU) during DNA replication. After mixing the two cell cultures that had been previously incorporated with CldU and IdU separately, the presence of these unusual deoxynucleosides in the genome can be detected by specific antibodies. For this, a DNA denaturation step is required to expose the sites of thymidine analogs incorporated. Subsequently, a secondary reaction using fluorochrome-labeled antibodies will generate distinct signals under fluorescence analysis. By using this method, DNA exchange verification (i.e., the presence of both CldU and IdU in the same cell) is possible using a standard fluorescence microscope. It typically takes 2-3 days from the thymidine analogs incorporation to results. Of note, ADExTA is relatively cheap and does not require transfections or harsh genetic manipulation. These features represent an advantage when compared to other time-consuming protocols that demand DNA manipulation to introduce distinct drug-resistance markers in different cells for posterior selection.

The recombinase Rad51 plays a key role in events of genetic exchange in Trypanosoma cruzi

Detection of genetic exchange has been a limiting factor to deepen the knowledge on the mechanisms by which Trypanosoma cruzi is able to generate progeny and genetic diversity. Here we show that incorporation of halogenated thymidine analogues, followed by immunostaining, is a reliable method not only to detect T. cruzi fused-cell hybrids, but also to quantify their percentage in populations of this parasite. Through this approach, we were able to detect and quantify fused-cell hybrids of T. cruzi clones CL Brener and Y. Given the increased detection of fused-cell hybrids in naturally-occurring hybrid CL Brener strain, which displays increased levels of RAD51 and BRCA2 transcripts, we further investigated the role of Rad51 – a recombinase involved in homologous recombination – in the process of genetic exchange. We also verified that the detection of fused-cell hybrids in T. cruzi overexpressing RAD51 is increased when compared to wild-type cells, suggesting a key role for Rad51 either in the formation or in the stabilization of fused-cell hybrids in this organism.

Characterization of Trypanosoma cruzi MutY DNA glycosylase ortholog and its role in oxidative stress response.

Trypanosoma cruzi is a protozoan parasite and the causative agent of Chagas disease. Like most living organisms, it is susceptible to oxidative stress, and must adapt to distinct environments. Hence, DNA repair is essential for its survival and the persistence of infection. Therefore, we studied whether T. cruzi has a homolog counterpart of the MutY enzyme (TcMYH), important in the DNA Base Excision Repair (BER) mechanism. Analysis of T. cruzi genome database showed that this parasite has a putative MutY DNA glycosylase sequence. We performed heterologous complementation assays using this genomic sequence. TcMYH complemented the Escherichia coli MutY- strain, reducing the mutation rate to a level similar to wild type. In in vitro assays, TcMYH was able to remove an adenine that was opposite to 8-oxoguanine. We have also constructed a T. cruzi lineage that overexpresses MYH. Although in standard conditions this lineage has similar growth to control cells, the overexpressor is more sensitive to hydrogen peroxide and glucose oxidase than the control, probably due to accumulation of AP sites in its DNA. Localization experiments with GFP-fused TcMYH showed this enzyme is present in both nucleus and mitochondrion. QPCR and MtOX results reinforce the presence and function of TcMYH in these two organelles. Our data suggest T. cruzi has a functional MYH DNA glycosylase, which participates in nuclear and mitochondrial DNA Base Excision Repair.

Oxidative stress and DNA lesions: the role of 8-oxoguanine lesions in Trypanosoma cruzi cell viability.

The main consequence of oxidative stress is the formation of DNA lesions, which can result in genomic instability and lead to cell death. Guanine is the base that is most susceptible to oxidation, due to its low redox potential, and 8-oxoguanine (8-oxoG) is the most common lesion. These characteristics make 8-oxoG a good cellular biomarker to indicate the extent of oxidative stress. If not repaired, 8-oxoG can pair with adenine and cause a G:C to T:A transversion. When 8-oxoG is inserted during DNA replication, it could generate double-strand breaks, which makes this lesion particularly deleterious. Trypanosoma cruzi needs to address various oxidative stress situations, such as the mammalian intracellular environment and the triatomine insect gut where it replicates. We focused on the MutT enzyme, which is responsible for removing 8-oxoG from the nucleotide pool. To investigate the importance of 8-oxoG during parasite infection of mammalian cells, we characterized the MutT gene in T. cruzi (TcMTH) and generated T. cruzi parasites heterologously expressing Escherichia coli MutT or overexpressing the TcMTH enzyme. In the epimastigote form, the recombinant and wild-type parasites displayed similar growth in normal conditions, but the MutT-expressing cells were more resistant to hydrogen peroxide treatment. The recombinant parasite also displayed significantly increased growth after 48 hours of infection in fibroblasts and macrophages when compared to wild-type cells, as well as increased parasitemia in Swiss mice. In addition, we demonstrated, using western blotting experiments, that MutT heterologous expression can influence the parasite antioxidant enzyme protein levels. These results indicate the importance of the 8-oxoG repair system for cell viability.