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
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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)