Trypanosoma brucei BRCA2 acts in a life cycle-specific genome stability process and dictates BRC repeat number-dependent RAD51 subnuclear dynamics

Nucleic Acids Research, Jan 2013

Trypanosoma brucei survives in mammals through antigenic variation, which is driven by RAD51-directed homologous recombination of Variant Surface Glycoproteins (VSG) genes, most of which reside in a subtelomeric repository of >1000 silent genes. A key regulator of RAD51 is BRCA2, which in T. brucei contains a dramatic expansion of a motif that mediates interaction with RAD51, termed the BRC repeats. BRCA2 mutants were made in both tsetse fly-derived and mammal-derived T. brucei, and we show that BRCA2 loss has less impact on the health of the former. In addition, we find that genome instability, a hallmark of BRCA2 loss in other organisms, is only seen in mammal-derived T. brucei. By generating cells expressing BRCA2 variants with altered BRC repeat numbers, we show that the BRC repeat expansion is crucial for RAD51 subnuclear dynamics after DNA damage. Finally, we document surprisingly limited co-localization of BRCA2 and RAD51 in the T. brucei nucleus, and we show that BRCA2 mutants display aberrant cell division, revealing a function distinct from BRC-mediated RAD51 interaction. We propose that BRCA2 acts to maintain the huge VSG repository of T. brucei, and this function has necessitated the evolution of extensive RAD51 interaction via the BRC repeats, allowing re-localization of the recombinase to general genome damage when needed.

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Trypanosoma brucei BRCA2 acts in a life cycle-specific genome stability process and dictates BRC repeat number-dependent RAD51 subnuclear dynamics

Anna Trenaman 1 2 Claire Hartley 1 2 Marko Prorocic 1 2 Danielle G. Passos-Silva 0 1 Moniek van den Hoek 1 2 Volodymyr Nechyporuk-Zloy 1 2 Carlos R. Machado 0 1 Richard McCulloch 1 2 0 Depto de Bioqumica, Universidade Federal de Minas Gerais , Avenida Ant onio Carlos, Caixa Postal 486, 30161-970 Belo Horizonte, MG, Brazil 1 Present address: Danielle G. Passos-Silva, Institute of Bioengineering , Ecole Polytechnique Fe d erale de Lausanne, Lausanne, Switzerland 2 The Wellcome Trust Centre for Molecular Parasitology, College of Medical Veterinary and Life Sciences, Institute of Infection, Immunity and Inflammation, University of Glasgow , Sir Graeme Davies Building, 120 University Place , Glasgow G12 8TA, UK - Trypanosoma brucei survives in mammals through antigenic variation, which is driven by RAD51directed homologous recombination of Variant Surface Glycoproteins (VSG) genes, most of which reside in a subtelomeric repository of >1000 silent genes. A key regulator of RAD51 is BRCA2, which in T. brucei contains a dramatic expansion of a motif that mediates interaction with RAD51, termed the BRC repeats. BRCA2 mutants were made in both tsetse fly-derived and mammal-derived T. brucei, and we show that BRCA2 loss has less impact on the health of the former. In addition, we find that genome instability, a hallmark of BRCA2 loss in other organisms, is only seen in mammal-derived T. brucei. By generating cells expressing BRCA2 variants with altered BRC repeat numbers, we show that the BRC repeat expansion is crucial for RAD51 subnuclear dynamics after DNA damage. Finally, we document surprisingly limited colocalization of BRCA2 and RAD51 in the T. brucei nucleus, and we show that BRCA2 mutants display aberrant cell division, revealing a function distinct from BRC-mediated RAD51 interaction. We propose that BRCA2 acts to maintain the huge VSG repository of T. brucei, and this function has necessitated the evolution of extensive RAD51 interaction via the BRC repeats, re-localization of the recombinase to genome damage when needed. African trypanosomes, such as Trypanosoma brucei, are protistan parasites that infect mammals in sub-Saharan Africa, where they are responsible for devastating disease in both humans and their domestic animals. Survival of T. brucei in mammals is dependent on antigenic variation, which involves switches in expression of variant surface glycoproteins (VSGs) that form a protective coat on the parasite cell surface (1,2). One VSG is expressed at a time, and continual switching to immunologically distinct VSGs allows part of the infecting population to survive successive waves of eradication by host immunity, prolonging the infection and enhancing transmission. VSG switching involves the activation of silent VSG genes by recombination, which results in copying the silent genes into specialized sites of transcription, termed VSG expression sites (3). VSGs are copied from an enormous (>1000 distinct genes) silent repository, mainly composed of subtelomeric VSG arrays. Most recombination-based VSG switching occurs through gene conversion reactions (2), which can take two forms: activation of functionally intact VSGs ( 5% of the repository) by whole gene conversion or activation of VSG pseudogenes ( 85% of the repository) by segmental gene conversion (2,4). Intact VSG gene conversion involves homologous recombination (HR), a universally conserved process that is critical in all organisms for reversing genotoxic damage and ensuring the completion of DNA replication (5). The key enzyme of eukaryotic HR is Rad51, which forms nucleoprotein filaments on singlestranded (ss) DNA at sites of damage and catalyses the transfer of the broken molecule to homologous sequences in an unbroken DNA molecule, leading to repair. Mutation of RAD51 in T. brucei impairs switching of intact VSGs (6), but the contribution of RAD51 and HR to segmental VSG conversion is unclear. Rad51 HR reactions are mediated by a number of proteins, which either directly influence Rad51 activity or act in upstream or downstream HR reaction steps (5). Trypanosoma brucei HR factors that perform both roles, such as RAD51 paralogues (7,8) and RMI1/TOPO3 (9,10), have been shown to act in VSG switching, reinforcing a close association between general HR and antigenic variation. BRCA2 has emerged as a key mediator of Rad51 function and is widely conserved, although not ubiquitous, in eukaryotes (11). BRCA2 orthologues vary considerably in size, from >3000 amino acids in mammals to proteins only 30 and 10% that size in Ustilago maydis (Brh2) (12) and Caenorhabditis elegans (CeBRC-2) (13), respectively. BRCA2 sequence conservation is limited out with two domains, termed the BRC repeats and the DSS1-DNA binding domain (DBD). BRC repeats mediate interaction with Rad51 during HR (14) and are a key conserved functional element, as each BRCA2 orthologue seems to retain at least one (11). In vertebrates, BRCA2 also binds Rad51 via an unrelated C-t (...truncated)


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Anna Trenaman, Claire Hartley, Marko Prorocic, Danielle G. Passos-Silva, Moniek van den Hoek, Volodymyr Nechyporuk-Zloy, Carlos R. Machado, Richard McCulloch. Trypanosoma brucei BRCA2 acts in a life cycle-specific genome stability process and dictates BRC repeat number-dependent RAD51 subnuclear dynamics, Nucleic Acids Research, 2013, pp. 943-960, 41/2, DOI: 10.1093/nar/gks1192