SummaryAfrican trypanosomes are unicellular parasites that use DNA recombination to evade the mammalian immune response. They do this in a process called antigenic variation, in which the parasites periodically switch the expression of VSG genes that encode distinct Variant Surface Glycoprotein coats. Recombination is used to move new VSG genes into specialised bloodstream VSG transcription sites. Genetic and molecular evidence has suggested that antigenic variation uses homologous recombination, but the detailed reaction pathways are not understood. In this study, we examine the recombination pathways used by trypanosomes to integrate transformed DNA into their genome, and show that they possess at least two pathways of homologous recombination. The primary mechanism is dependent upon RAD51, but a subsidiary pathway exists that is RAD51-independent. Both pathways contribute to antigenic variation. We show that the RAD51-independent pathway is capable of recombining DNA substrates with very short lengths of sequence homology and in some cases aberrant recombination reactions can be detected using such microhomologies.

Site-specific recombinases of the integrase family usually require cofactors to impart directionality in the recombination reactions that they catalyze. The FimB integrase inverts the Escherichia coli fim switch (fimS) in the on-to-off and off-to-on directions with approximately equal efficiency. Inhibiting DNA gyrase with novobiocin caused inversion to become biased in the off-to-on direction. This directionality was not due to differential DNA topological distortion of fimS in the on and off phases by the activity of its resident P fimA promoter. Instead, the leucine-responsive regulatory (Lrp) protein was found to determine switching outcomes. Knocking out the lrp gene or abolishing Lrp binding sites 1 and 2 within fimS completely reversed the response of the switch to DNA relaxation. Inactivation of either Lrp site alone resulted in mild on-to-off bias, showing that they act together to influence the response of the switch to changes in DNA supercoiling. Thus, Lrp is not merely an architectural element organizing the fim invertasome, it collaborates with DNA supercoiling to determine the directionality of the DNA inversion event.Site-specific recombinases of the integrase family are usually associated with the integration and excision of DNA sequences such as bacteriophage genomes from bacterial chromosomes or other replicons. The best-studied integrase is Int, the prototypic member of the family that catalyzes the integration and excision of bacteriophage lambda from the chromosome of Escherichia coli (33,40). Although the integration and excision reactions both require Int, an additional phage-encoded factor called the excisionase (Xis) confers directionality by being specific for the excision reaction. Despite its name, the excisionase has no enzymatic activity. Instead, it is an architectural element that helps to organize the local structure of lambda DNA in a way that favors the excision reaction. The Xis protein has been classified as a recombination directionality factor (RDF), and several other proteins have been identified that provide, or may provide, an analogous function in other integrase-dependent site-specific recombination reactions (23-25). The requirement for the RDFs arises due to the similarities of the DNA substrates and products of the integration and excision reactions. The RDF confers directionality by stimulating one reaction while inhibiting the other.An integrase-mediated site-specific recombination event controls the phase-variable expression of type 1 fimbriae in E. coli. A key difference between the fimbrial and phage recombination mechanisms is that the fimbrial system involves DNA inversion and not integration/excision. The promoter for fim operon transcription (P fimA ) is carried on a 314-bp invertible DNA element called the fim switch (fimS), and expression of the fim structural genes depends on its orientation (1, 15). With P fimA directed toward the fim operon, the genes are transcribed, and when it is inverted to the opposite orientation, the fim operon is silent (Fig. ...

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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.