RESUMEN
RNA-DNA hybrids are epigenetic features of all genomes that intersect with many processes, including transcription, telomere homeostasis, and centromere function. Increasing evidence suggests that RNA-DNA hybrids can provide two conflicting roles in the maintenance and transmission of genomes: They can be the triggers of DNA damage, leading to genome change, or can aid the DNA repair processes needed to respond to DNA lesions. Evasion of host immunity by African trypanosomes, such as Trypanosoma brucei, relies on targeted recombination of silent Variant Surface Glycoprotein (VSG) genes into a specialized telomeric locus that directs transcription of just one VSG from thousands. How such VSG recombination is targeted and initiated is unclear. Here, we show that a key enzyme of T. brucei homologous recombination, RAD51, interacts with RNA-DNA hybrids. In addition, we show that RNA-DNA hybrids display a genome-wide colocalization with DNA breaks and that this relationship is impaired by mutation of RAD51. Finally, we show that RAD51 acts to repair highly abundant, localised DNA breaks at the single transcribed VSG and that mutation of RAD51 alters RNA-DNA hybrid abundance at 70 bp repeats both around the transcribed VSG and across the silent VSG archive. This work reveals a widespread, generalised role for RNA-DNA hybrids in directing RAD51 activity during recombination and uncovers a specialised application of this interplay during targeted DNA break repair needed for the critical T. brucei immune evasion reaction of antigenic variation.
Asunto(s)
Trypanosoma brucei brucei , Estructuras R-Loop , Variación Antigénica/genética , Roturas del ADN , ADN , ARN , Glicoproteínas Variantes de Superficie de Trypanosoma/genéticaRESUMEN
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. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.
Asunto(s)
Recombinación Homóloga/genética , Leishmania major/genética , Leishmaniasis Cutánea/genética , Recombinasa Rad51/genética , Sistemas CRISPR-Cas/genética , Daño del ADN/genética , Reparación del ADN/genética , Replicación del ADN/genética , Técnicas de Inactivación de Genes , Genoma/genética , Humanos , Leishmania major/patogenicidad , Leishmaniasis Cutánea/parasitologíaRESUMEN
Ribonucleotides represent a threat to DNA genome stability and transmission. Two types of Ribonuclease H (RNase H) excise ribonucleotides when they form part of the DNA strand, or hydrolyse RNA when it base-pairs with DNA in structures termed R-loops. Loss of either RNase H is lethal in mammals, whereas yeast survives the absence of both enzymes. RNase H1 loss is tolerated by the parasite Trypanosoma brucei but no work has examined the function of RNase H2. Here we show that loss of T. brucei RNase H2 (TbRH2A) leads to growth and cell cycle arrest that is concomitant with accumulation of nuclear damage at sites of RNA polymerase (Pol) II transcription initiation, revealing a novel and critical role for RNase H2. Differential gene expression analysis reveals limited overall changes in RNA levels for RNA Pol II genes after TbRH2A loss, but increased perturbation of nucleotide metabolic genes. Finally, we show that TbRH2A loss causes R-loop and DNA damage accumulation in telomeric RNA Pol I transcription sites, also leading to altered gene expression. Thus, we demonstrate separation of function between two nuclear T. brucei RNase H enzymes during RNA Pol II transcription, but overlap in function during RNA Pol I-mediated gene expression during host immune evasion.
Asunto(s)
Antígenos de Protozoos/genética , Inestabilidad Genómica/genética , Ribonucleasa H/genética , Iniciación de la Transcripción Genética , Animales , Antígenos de Protozoos/inmunología , ADN/química , ADN/genética , Daño del ADN/genética , Replicación del ADN/genética , Regulación de la Expresión Génica/genética , Humanos , Conformación de Ácido Nucleico , ARN/química , ARN/genética , ARN Polimerasa I/genética , ARN Polimerasa II/genética , Ribonucleasa H/química , Ribonucleasa H/inmunología , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/inmunología , Trypanosoma brucei brucei/patogenicidadRESUMEN
Switching of the Variant Surface Glycoprotein (VSG) in Trypanosoma brucei provides a crucial host immune evasion strategy that is catalysed both by transcription and recombination reactions, each operating within specialised telomeric VSG expression sites (ES). VSG switching is likely triggered by events focused on the single actively transcribed ES, from a repertoire of around 15, but the nature of such events is unclear. Here we show that RNA-DNA hybrids, called R-loops, form preferentially within sequences termed the 70 bp repeats in the actively transcribed ES, but spread throughout the active and inactive ES, in the absence of RNase H1, which degrades R-loops. Loss of RNase H1 also leads to increased levels of VSG coat switching and replication-associated genome damage, some of which accumulates within the active ES. This work indicates VSG ES architecture elicits R-loop formation, and that these RNA-DNA hybrids connect T. brucei immune evasion by transcription and recombination.
Asunto(s)
Evasión Inmune/genética , Ribonucleasa H/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/inmunología , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/inmunología , Animales , Variación Antigénica , Daño del ADN , Genoma de Protozoos , Interacciones Huésped-Parásitos/genética , Interacciones Huésped-Parásitos/inmunología , Humanos , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Ribonucleasa H/deficiencia , Trypanosoma brucei brucei/patogenicidad , Tripanosomiasis Africana/inmunología , Tripanosomiasis Africana/parasitologíaRESUMEN
R-loops are stable RNA-DNA hybrids that have been implicated in transcription initiation and termination, as well as in telomere maintenance, chromatin formation, and genome replication and instability. RNA Polymerase (Pol) II transcription in the protozoan parasite Trypanosoma brucei is highly unusual: virtually all genes are co-transcribed from multigene transcription units, with mRNAs generated by linked trans-splicing and polyadenylation, and transcription initiation sites display no conserved promoter motifs. Here, we describe the genome-wide distribution of R-loops in wild type mammal-infective T. brucei and in mutants lacking RNase H1, revealing both conserved and diverged functions. Conserved localization was found at centromeres, rRNA genes and retrotransposon-associated genes. RNA Pol II transcription initiation sites also displayed R-loops, suggesting a broadly conserved role despite the lack of promoter conservation or transcription initiation regulation. However, the most abundant sites of R-loop enrichment were within the regions between coding sequences of the multigene transcription units, where the hybrids coincide with sites of polyadenylation and nucleosome-depletion. Thus, instead of functioning in transcription termination the most widespread localization of R-loops in T. brucei suggests a novel correlation with pre-mRNA processing. Finally, we find little evidence for correlation between R-loop localization and mapped sites of DNA replication initiation.
Asunto(s)
Genoma de Protozoos , Mutación , Trypanosoma brucei brucei/genética , Sitios de Unión , Centrómero , Cromatina/química , Regulación de la Expresión Génica , Nucleosomas , Poliadenilación , Regiones Promotoras Genéticas , Dominios Proteicos , Proteínas Protozoarias/genética , ARN Polimerasa II/metabolismo , ARN Ribosómico/química , Sitio de Iniciación de la Transcripción , Transcripción Genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genéticaRESUMEN
Homologous recombination in Trypanosoma brucei is used for moving variant surface glycoprotein (VSG) genes into expression sites during immune evasion by antigenic variation. A major route for such VSG switching is gene conversion reactions in which RAD51, a universally conserved recombinase, catalyses homology-directed strand exchange. In any eukaryote, RAD51-directed strand exchange in vivo is mediated by further factors, including RAD51-related proteins termed Rad51 paralogues. These appear to be ubiquitously conserved, although their detailed roles in recombination remain unclear. In T. brucei, four putative RAD51 paralogue genes have been identified by sequence homology. Here we show that all four RAD51 paralogues act in DNA repair, recombination and RAD51 subnuclear dynamics, though not equivalently, while mutation of only one RAD51 paralogue gene significantly impedes VSG switching. We also show that the T. brucei RAD51 paralogues interact, and that the complexes they form may explain the distinct phenotypes of the mutants as well as observed expression interdependency. Finally, we document the Rad51 paralogues that are encoded by a wide range of protists, demonstrating that the Rad51 paralogue repertoire in T. brucei is unusually large among microbial eukaryotes and that one member of the protein family corresponds with a key, conserved eukaryotic Rad51 paralogue.
Asunto(s)
Variación Antigénica , Antígenos de Protozoos/metabolismo , Reparación del ADN , Mapeo de Interacción de Proteínas , Proteínas Protozoarias/metabolismo , Recombinasa Rad51/metabolismo , Trypanosoma brucei brucei/fisiología , Antígenos de Protozoos/genética , Secuencia Conservada , Eliminación de Gen , Proteínas Protozoarias/genética , Recombinasa Rad51/genética , Recombinación Genética , Homología de Secuencia de Aminoácido , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/inmunologíaRESUMEN
Trypanosoma brucei evades mammalian immunity by using recombination to switch its surface-expressed variant surface glycoprotein (VSG), while ensuring that only one of many subtelomeric multigene VSG expression sites are transcribed at a time. DNA repair activities have been implicated in the catalysis of VSG switching by recombination, not transcriptional control. How VSG switching is signaled to guide the appropriate reaction or to integrate switching into parasite growth is unknown. Here, we show that the loss of ATR, a DNA damage-signaling protein kinase, is lethal, causing nuclear genome instability and increased VSG switching through VSG-localized damage. Furthermore, ATR loss leads to the increased transcription of silent VSG expression sites and expression of mixed VSGs on the cell surface, effects that are associated with the altered localization of RNA polymerase I and VEX1. This work shows that ATR acts in antigenic variation both through DNA damage signaling and surface antigen expression control.
Asunto(s)
Variación Antigénica , Antígenos de Superficie/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Daño del ADN , ARN Polimerasa I/metabolismo , Transcripción Genética , Trypanosoma brucei brucei/enzimología , Alelos , Núcleo Celular/patología , Proliferación Celular , Supervivencia Celular , Regulación de la Expresión Génica , Genoma , Modelos Biológicos , Transporte de Proteínas , Proteínas Protozoarias/metabolismo , Interferencia de ARN , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/genéticaRESUMEN
DNA replication is needed to duplicate a cell's genome in S phase and segregate it during cell division. Previous work in Leishmania detected DNA replication initiation at just a single region in each chromosome, an organisation predicted to be insufficient for complete genome duplication within S phase. Here, we show that acetylated histone H3 (AcH3), base J and a kinetochore factor co-localise in each chromosome at only a single locus, which corresponds with previously mapped DNA replication initiation regions and is demarcated by localised G/T skew and G4 patterns. In addition, we describe previously undetected subtelomeric DNA replication in G2/M and G1-phase-enriched cells. Finally, we show that subtelomeric DNA replication, unlike chromosome-internal DNA replication, is sensitive to hydroxyurea and dependent on 9-1-1 activity. These findings indicate that Leishmania's genome duplication programme employs subtelomeric DNA replication initiation, possibly extending beyond S phase, to support predominantly chromosome-internal DNA replication initiation within S phase.
Asunto(s)
Estructuras Cromosómicas , Replicación del ADN/genética , Duplicación de Gen/genética , Genoma de Protozoos/genética , Leishmania major/genética , Estructuras Cromosómicas/química , Estructuras Cromosómicas/genética , Estructuras Cromosómicas/metabolismo , Cromosomas/química , Cromosomas/genética , Histonas/genética , Histonas/metabolismo , Fase S/genéticaRESUMEN
Intergenerational effects arise when parents' actions influence the reproduction and survival of their offspring and possibly later descendants. Models suggest that intergenerational effects have important implications for both population dynamical patterns and the evolution of life-history traits. However, these will depend on the nature and duration of intergenerational effects. Here we show that manipulating parental food environments of soil mites produced intergenerational effects that were still detectable in the life histories of descendents three generations later. Intergenerational effects varied in different environments and from one generation to the next. In low-food environments, variation in egg size altered a trade-off between age and size at maturity and had little effect on the size of eggs produced in subsequent generations. Consequently, intergenerational effects decreased over time. In contrast, in high-food environments, variation in egg size predominantly influenced a trade-off between fecundity and adult survival and generated increasing variation in egg size. As a result, the persistence and significance of intergenerational effects varied between high- and low-food environments. Context-dependent intergenerational effects can therefore have complex but important effects on population dynamics.
Asunto(s)
Acaridae/fisiología , Ambiente , Acaridae/anatomía & histología , Factores de Edad , Animales , Conducta Alimentaria , Femenino , Masculino , Óvulo/citología , Dinámica Poblacional , ReproducciónRESUMEN
The well studied trade-off between offspring size and offspring number assumes that offspring fitness increases with increasing per-offspring investment. Where mothers differ genetically or exhibit plastic variation in reproductive effort, there can be variation in per capita investment in offspring, and via this trade-off, variation in fecundity. Variation in per capita investment will affect juvenile performance directly--a classical maternal effect--while variation in fecundity will also affect offspring performance by altering the offsprings' competitive environment. The importance of this trade-off, while a focus of evolutionary research, is not often considered in discussions about population dynamics. Here, we use a factorial experiment to determine what proportion of variation in offspring performance can be ascribed to maternal effects and what proportion to the competitive environment linked to the size-number trade-off. Our results suggest that classical maternal effects are significant, but that in our system, the competitive environment, which is linked to maternal environments by fecundity, can be a far more substantial influence.
Asunto(s)
Ácaros y Garrapatas/fisiología , Ácaros y Garrapatas/crecimiento & desarrollo , Animales , Tamaño de la Nidada , Femenino , Modelos Lineales , Masculino , Conducta Materna , Dinámica Poblacional , Distribución Aleatoria , Análisis de RegresiónRESUMEN
Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating - a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility.
Asunto(s)
Variación Antigénica , Replicación del ADN , Telómero/metabolismo , Transcripción Genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superficie de Trypanosoma/biosíntesis , Roturas del ADN , Reparación del ADN , RecQ Helicasas/metabolismoRESUMEN
In most organisms, transitions between different life-history stages occur later and at smaller sizes as growth conditions deteriorate. Day and Rowe recently proposed that this pattern could be explained by the existence of developmental thresholds (minimum sizes or levels of condition below which transitions are unable to proceed). The developmental-threshold model predicts that the reaction norm of age and size at maturity will rotate in an anticlockwise manner from positive to a shallow negative slope if: (i) initial body size or condition is reduced; and/or (ii) some individuals encounter poor growth conditions at increasingly early developmental stages. We tested these predictions by rearing replicated populations of soil mites Sancassania berlesei (Michael) under different growth conditions. High-food environments produced a vertical relationship between age and size at maturity. The slope became increasingly shallow as food was reduced. By contrast, high food in the maternal environment reduced the slope of the reaction norm of age and size at maturity, whereas low food increased it. Overall, the reaction norm of age and size at maturity in S. berlesei was significantly nonlinear and differed for males and females. We describe how growth conditions, mother's environment and sex determine age and size at maturity in S. berlesei.
Asunto(s)
Acaridae/crecimiento & desarrollo , Constitución Corporal/fisiología , Ambiente , Modelos Biológicos , Maduración Sexual/fisiología , Acaridae/fisiología , Factores de Edad , Animales , Femenino , Masculino , Factores SexualesRESUMEN
In variable environments, it is probable that environmental conditions in the past can influence demographic performance now. Cohort effects occur when these delayed life-history effects are synchronized among groups of individuals in a population. Here we show how plasticity in density-dependent demographic traits throughout the life cycle can lead to cohort effects and that there can be substantial population dynamic consequences of these effects. We show experimentally that density and food conditions early in development can influence subsequent juvenile life-history traits. We also show that conditions early in development can interact with conditions at maturity to shape future adult performance. In fact, conditions such as food availability and density at maturity, like conditions early in development, can generate cohort effects in mature stages. Based on these data, and on current theory about the effects of plasticity generated by historical environments, we make predictions about the consequences of such changes on density-dependent demography and on mite population dynamics. We use a stochastic cohort effects model to generate a range of population dynamics. In accordance with the theory, we find the predicted changes in the strength of density dependence and associated changes in population dynamics and population variability.