Inactivation of Mre11 Does Not Affect VSG Gene Duplication Mediated by Homologous Recombination in Trypanosoma brucei

Abstract

We demonstrate, by gene deletion analysis, that Mre11 has a critical role in maintaining genomic integrity in Trypanosoma brucei. mre11 ؊/؊ null mutant strains exhibited retarded growth but no delay or disruption of cell cycle progression. They showed also a weak hyporecombination phenotype and the accumulation of gross chromosomal rearrangements, which did not involve sequence translocation, telomere loss, or formation of new telomeres. The trypanosome mre11 ؊/؊ strains were hypersensitive to phleomycin, a mutagen causing DNA double strand breaks (DSBs) but, in contrast to mre11 ؊/؊ null mutants in other organisms and T. brucei rad51 ؊/؊ null mutants, displayed no hypersensitivity to methyl methanesulfonate, which causes point mutations and DSBs. Mre11 therefore is important for the repair of chromosomal damage and DSBs in trypanosomes, although in this organism the intersection of repair pathways appears to differ from that in other organisms. Mre11 inactivation appears not to affect VSG gene switching during antigenic variation of a laboratory strain, which is perhaps surprising given the importance of homologous recombination during this process.

Mismatch Repair Regulates Homologous Recombination, but Has Little Influence on Antigenic Variation, in Trypanosoma brucei

“…MSH2 and MLH1 mutants for assaying VSG switching were made in strain 3174, 2 a derivative of MITat1.2a (47). Determination of VSG switching frequency and pattern has been described previously (16,19,20). To assay the effect of base mismatches on recombination, MITat1.2a cells were transformed with the construct pTHT (43,44); targeting of the HYG gene (see text) to the tubulin array was confirmed by Southern mapping with KpnI-or EcoRI-digested genomic DNA (data not shown).…”

“…Consistent with this genetic analysis, inactivation of KU70 or KU80, which catalyze a non-homologous end-joining pathway of DNA repair in other organisms (17,18), does not affect VSG switching (19). Surprisingly, mutation of MRE11 also does not affect VSG switching (20), despite this gene encoding an enzyme that has been proposed to have many roles in homologous and non-homologous recombination (21).…”

DNA Recombination Strategies During Antigenic Variation in the African Trypanosome

“…Moreover, though a later study confirmed that ISceI-mediated DSB formation can activate switching in a BES location-dependent fashion, strict localization of breaks within the active BES and around the 70-bp repeats was not confirmed, and instead ligation-mediated PCR suggested that all BES are fragile adjacent to the telomere (128). Finally, as noted above, the lack of a role for MRE11 in VSG switching is perplexing, given its central function in DSB detection and repair (152). One explanation for these inconsistencies, as we have argued throughout, is that VSG recombination cannot be explained by a single mechanism, or by a sequence to direct that mechanism, and the system acts flexibly through several routes.…”

“…Understanding where this process occurs would be very revealing: are the array VSG pseudogenes "assembly intermediates" that arise from ongoing subtelomeric recombination, or might the reaction be rapid enough to allow assembly within the active BES without leading to protracted expression of nonfunctional VSG protein (87)? Loss of array VSGs during growth of T. brucei BRCA2 and MRE11 mutants hints at recombination within the subtelomeres, but the nature and scale of such events is unclear (109,137,152).…”

“…5), composed of MRE11, RAD50 and Xrs2/NBS1 in eukaryotes, with roles in processing DSBs, activating the DNA damage response through the kinase ATM, bridging DNA molecules and suppressing non-HR repair (151). MRE11 mutants in T. brucei display DNA damage repair and genome instability phenotypes (152,153), including gross chromosomal rearrangements due to loss of subtelomeric VSGs, an instability also seen in bloodstream form T. brucei BRCA2 mutants (109,137). Despite this, loss of MRE11 has no detectable effect on VSG switching, questioning whether DSBs can initiate the reaction (see below) or whether loss of MRE11 alleviates suppression of alternative DSB repair reactions that can be upregulated and affect switching.…”