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

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

Zinc-finger recombinase activities in vitro

Zinc-finger recombinases (ZFRs) are chimaeric proteins comprising a serine recombinase catalytic domain linked to a zinc-finger DNA binding domain. ZFRs can be tailored to promote site-specific recombination at diverse ‘Z-sites’, which each comprise a central core sequence flanked by zinc-finger domain-binding motifs. Here, we show that purified ZFRs catalyse efficient high-specificity reciprocal recombination between pairs of Z-sites in vitro. No off-site activity was detected. Under different reaction conditions, ZFRs can catalyse Z-site-specific double-strand DNA cleavage. ZFR recombination activity in Escherichia coli and in vitro is highly dependent on the length of the Z-site core sequence. We show that this length effect is manifested at reaction steps prior to formation of recombinants (binding, synapsis and DNA cleavage). The design of the ZFR protein itself is also a crucial variable affecting activity. A ZFR with a very short (2 amino acids) peptide linkage between the catalytic and zinc-finger domains has high activity in vitro, whereas a ZFR with a very long linker was less recombination-proficient and less sensitive to variations in Z-site length. We discuss the causes of these phenomena, and their implications for practical applications of ZFRs

Sequence selectivity of the resolvase catalytic domain: implications for Z-resolvase design

The extent of sequence specificity of the Tn3 resolvase catalytic domain was investigated by creating libraries of Tn3 site I variants in which all of the central 16 bp were systematically randomised in overlapping 4 bp blocks and recombination deficient and recombination proficient site I variants were selected using two different independent selection strategies employing an activated Tn3 resolvase mutant NM. A degree of flexibility in the sequences permitted in the central 16 bp of the Tn3 site I was observed especially at the positions 4, 7 and 8, but accumulating changes was found to be in general detrimental to recombination. The data was compared to the naturally occurring site I sequences associated with proteins from the Tn3 resolvase family, and integrated with the available structural information revealing a number of residues in the extended arm region that could account for the sequence selectivity observed. The sequence selectivity of the activated Tn3 resolvase NM catalytic domain was tested in the Z-resolvase context employing a similar but less exhaustive selection strategy using a purified Z-resolvase Z-R(NM). Z-resolvases with sequence selectivity that is different to that of Z-R(NM) were constructed using catalytic domains of activated mutants of Sin and Tn21 resolvases and their in vivo and in vitro properties were tested, highlighting the universality of the Z-resolvase approach and its potential for the future applications. A number of issues concerning the Z-resolvase design such as the optimum length of Z-sites, what is the effect of the Zif268 DNA-binding domain on catalytic activity i.e. is it activating or inhibiting, is symmetry a prerequisite in the design of Z-sites or can a Z-resolvase catalyse recombination on sites with an odd number of bases between Zif268 binding sites i.e. one half-site longer than the other, what is the relative influence of the Z-resolvase linker length, and can Z-resolvase be complemented by resolvase and catalyse recombination on appropriately designed hybrid sites were explored. The sequence selectivity of catalytic domains of Sin and Tn21 resolvases was compared using a combination of a mutant library selection strategy and the Sin-Tn21 resolvase hybrid experiments. An attempt to change the sequence selectivity of Tn3 resolvase catalytic domain into that of Sin resolvase, both in the resolvase and Z-resolvase context by mutating the specific residues, implicated in catalytic domain sequence selectivity was performed. The sequence selectivity of activated Tn3 resolvase catalytic domain was successfully changed into that of Sin resolvas