An endonuclease-generated DNA break induces antigenic switching in Trypanosoma brucei

Laboratory of Lymphocyte Biology, The Rockefeller University, New York, New York 10065, USA.
Nature (Impact Factor: 41.46). 05/2009; 459(7244):278-81. DOI: 10.1038/nature07982
Source: PubMed


Trypanosoma brucei is the causative agent of African sleeping sickness in humans and one of the causes of nagana in cattle. This protozoan parasite evades the host immune system by antigenic variation, a periodic switching of its variant surface glycoprotein (VSG) coat. VSG switching is spontaneous and occurs at a rate of about 10(-2)-10(-3) per population doubling in recent isolates from nature, but at a markedly reduced rate (10(-5)-10(-6)) in laboratory-adapted strains. VSG switching is thought to occur predominantly through gene conversion, a form of homologous recombination initiated by a DNA lesion that is used by other pathogens (for example, Candida albicans, Borrelia sp. and Neisseria gonorrhoeae) to generate surface protein diversity, and by B lymphocytes of the vertebrate immune system to generate antibody diversity. Very little is known about the molecular mechanism of VSG switching in T. brucei. Here we demonstrate that the introduction of a DNA double-stranded break (DSB) adjacent to the approximately 70-base-pair (bp) repeats upstream of the transcribed VSG gene increases switching in vitro approximately 250-fold, producing switched clones with a frequency and features similar to those generated early in an infection. We were also able to detect spontaneous DSBs within the 70-bp repeats upstream of the actively transcribed VSG gene, indicating that a DSB is a natural intermediate of VSG gene conversion and that VSG switching is the result of the resolution of this DSB by break-induced replication.

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Available from: Oliver Dreesen, Apr 16, 2014
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    • "A recent study of African trypanosome species, the first identified and best understood model of protozoan antigenic variation, has shown that DNA helix-destabilizing TAA:TTA repeats within the VSG antigen genes induce antigenic variation and VSG sequence diversification through a recombination pathway that shares some resemblance to the mechanism of var gene diversification outlined here (61). Both the VSG and var multi-gene families evolve by ectopic recombination events through a mechanism of break-induced replication (62,63), but the inducing DNA structures appear to be different in the two parasites. Where specific TAA:TTA repeats destabilize the DNA helix of trypanosome VSG genes (64), Plasmodium var gene DSS are formed from diverse sequences and are predicted to form stable secondary structures. "
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    ABSTRACT: Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite's sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens.
    Full-text · Article · Nov 2013 · Nucleic Acids Research
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    • "Other studies also suggest that DNA replication acts in antigenic variation. The DNA DSBs found within the VSG BESs (Boothroyd et al., 2009; Glover et al., 2013) may form following replication stalling and replication-fork collapse . In addition, inheritance of the active and silent VSGs in their previously transcribed or silent states clearly depends upon the replication process. "
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    ABSTRACT: African trypanosomes are lethal human and animal parasites that use antigenic variation for evasion of host adaptive immunity. To facilitate antigenic variation, trypanosomes dedicate approximately one third of their nuclear genome, including many minichromosomes, and possibly all sub-telomeres, to variant surface glycoprotein (VSG) genes and associated sequences. Antigenic variation requires transcription of a single VSG by RNA polymerase I (Pol-I), with silencing of other VSGs, and periodic switching of the expressed gene, typically via DNA recombination with duplicative translocation of a new VSG to the active site. Thus, telomeric location, epigenetic controls and monoallelic transcription by Pol-I at an extranucleolar site are prominent features of VSGs and their expression, with telomeres, chromatin structure and nuclear organisation all making vitally important contributions to monoallelic VSG expression control and switching. We discuss VSG transcription, recombination and replication control within this chromosomal and sub-nuclear context.
    Full-text · Article · Sep 2013 · Cellular Microbiology
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    • "Although VSGs are highly immunogenic, T. brucei manages to escape the immune response by switching the expression of VSG up to once per 100 cell divisions [120–122]. This VSG switching often involves gene conversion of VSG cassettes into the active ES, creating and expressing a novel VSG gene that has not previously been seen by the immune system [123,124]. This intricate monoallelic expression and periodical switching of VSGs enable the parasites to evade the host immune response (reviewed in [125–127]), and it is thus difficult to develop effective vaccines. "
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    ABSTRACT: Faithful transmission of genetic material is essential for the survival of all organisms. Eukaryotic chromosome segregation is driven by the kinetochore that assembles onto centromeric DNA to capture spindle microtubules and govern the movement of chromosomes. Its molecular mechanism has been actively studied in conventional model eukaryotes, such as yeasts, worms, flies and human. However, these organisms are closely related in the evolutionary time scale and it therefore remains unclear whether all eukaryotes use a similar mechanism. The evolutionary origins of the segregation apparatus also remain enigmatic. To gain insights into these questions, it is critical to perform comparative studies. Here, we review our current understanding of the mitotic mechanism in Trypanosoma brucei, an experimentally tractable kinetoplastid parasite that branched early in eukaryotic history. No canonical kinetochore component has been identified, and the design principle of kinetochores might be fundamentally different in kinetoplastids. Furthermore, these organisms do not appear to possess a functional spindle checkpoint that monitors kinetochore-microtubule attachments. With these unique features and the long evolutionary distance from other eukaryotes, understanding the mechanism of chromosome segregation in T. brucei should reveal fundamental requirements for the eukaryotic segregation machinery, and may also provide hints about the origin and evolution of the segregation apparatus.
    Full-text · Article · May 2013 · Open Biology
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