Richard McCulloch

Two related recombinases are required for site-specific recombination at dif and cer in E. coli K12.

The stable inheritance of ColE1-related plasmids and the normal partition of the E. coli chromosome require the function of the Xer site-specific recombination system. We show that in addition to the XerC recombinase, whose function has already been implicated in this system, a second chromosomally encoded recombinase, XerD, is required. The XerC and XerD proteins show 37% identity and bind to separate halves of the recombination site. Both proteins act catalytically in the recombination reaction. Recombination site asymmetry and the requirement of two recombinases ensure that only correctly aligned sites are recombined. We predict that normal partition of most circular chromosomes requires the participation of site-specific recombination to convert any multimers (arising by homologous recombination) to monomers.

Why are parasite contingency genes often associated with telomeres

Contingency genes are common in pathogenic microbes and enable, through pre-emptive mutational events, rapid, clonal switches in phenotype that are conducive to survival and proliferation in hosts. Antigenic variation, which is a highly successful survival strategy employed by eubacterial and eukaryotic pathogens, involves large repertoires of distinct contingency genes that are expressed differentially, enabling evasion of host acquired immunity. Most, but not all, antigenic variation systems make extensive use of subtelomeres. Study of model systems has shown that subtelomeres have unusual properties, including reversible silencing of genes mediated by proteins binding to the telomere, and engagement in ectopic recombination with other subtelomeres. There is a general theory that subtelomeric location confers a capacity for gene diversification through such recombination, although experimental evidence is that there is no increased mitotic recombination at such loci and that sequence homogenisation occurs. Possible benefits of subtelomeric location for pathogen contingency systems are reversible gene silencing, which could contribute to systems for gene switching and mutually exclusive expression, and ectopic recombination, leading to gene family diversification. We examine, in several antigenic variation systems, what possible benefits apply.

Molecular mechanisms underlying the control of antigenic variation in African trypanosomes

African trypanosomes escape the host adaptive immune response by switching their dense protective coat of Variant Surface Glycoprotein (VSG). Each cell expresses only one VSG gene at a time from a telomeric expression site (ES). The ‘pre-genomic’ era saw the identification of the range of pathways involving VSG recombination in the context of mono-telomeric VSG transcription. A prominent feature of the early post-genomic era is the description of the molecular machineries involved in these processes. We describe the factors and sequences recently linked to mutually exclusive transcription and VSG recombination, and how these act in the control of the key virulence mechanism of antigenic variation.

An update on antigenic variation in African trypanosomes

African trypanosomes can spend a long time in the blood of their mammalian host, where they are exposed to the immune system and are thought to take advantage of it to modulate their own numbers. Their major immunogenic protein is the variant surface glycoprotein (VSG), the gene for which must be in one of the 20--40 specialized telomeric expression sites in order to be transcribed. Trypanosomes escape antibody-mediated destruction through periodic changes of the expressed VSG gene from a repertoire of approximately 1000. How do trypanosomes exclusively express only one VSG and how do they switch between them?

Antigenic variation in the African trypanosome: molecular mechanisms and phenotypic complexity

Antigenic variation is an immune evasion strategy that has evolved in viral, bacterial and protistan pathogens. In the African trypanosome this involves stochastic switches in the composition of a variant surface glycoprotein (VSG) coat, using a massive archive of silent VSG genes to change the identity of the single VSG expressed at a time. VSG switching is driven primarily by recombination reactions that move silent VSGs into specialized expression sites, though transcription‐based switching can also occur. Here we discuss what is being revealed about the machinery that underlies these switching mechanisms, including what parallels can be drawn with other pathogens. In addition, we discuss how such switching reactions act in a hierarchy and contribute to pathogen survival in the face of immune attack, including the establishment and maintenance of chronic infections, leading to host–host transmission.

Transformation of Monomorphic and Pleomorphic Trypanosoma brucei

African trypanosomes, such as Trypanosoma brucei, are protozoan parasites of mammals that were first described over 100 hundred years ago. They have long been the subjects of biological investigation, which has yielded insights into a number of fundamental, as well as novel, cellular processes in all organisms. In the last decade or so, genetic manipulation of trypanosomes has become possible through DNA transformation, allowing yet more detailed analysis of the biology of the parasite. One facet of this is that DNA transformation has itself been used as an assay for recombination and will undoubtedly lead to further genetic approaches to examine this process. Here we describe protocols for DNA transformation of Trypanosoma brucei, including two different life cycle stages and two different strain types that are distinguished by morphological and developmental criteria. We consider the application of transformation to recombination, as well as the uses of transforming the different life cycle stages and strain types.

Sequence homology and microhomology dominate chromosomal double-strand break repair in African trypanosomes

Genetic diversity in fungi and mammals is generated through mitotic double-strand break-repair (DSBR), typically involving homologous recombination (HR) or non-homologous end joining (NHEJ). Microhomology-mediated joining appears to serve a subsidiary function. The African trypanosome, a divergent protozoan parasite, relies upon rearrangement of subtelomeric variant surface glycoprotein (VSG) genes to achieve antigenic variation. Evidence suggests an absence of NHEJ but chromosomal repair remains largely unexplored. We used a system based on I-SceI meganuclease and monitored temporally constrained DSBR at a specific chromosomal site in bloodstream form Trypanosoma brucei. In response to the lesion, adjacent single-stranded DNA was generated; the homologous strand-exchange factor, Rad51, accumulated into foci; a G2M checkpoint was activated and >50% of cells displayed successful repair. Quantitative analysis of DSBR pathways employed indicated that inter-chromosomal HR dominated. HR displayed a strong preference for the allelic template but also the capacity to interact with homologous sequence on heterologous chromosomes. Intra-chromosomal joining was predominantly, and possibly exclusively, microhomology mediated, a situation unique among organisms examined to date. These DSBR pathways available to T. brucei likely underlie patterns of antigenic variation and the evolution of the vast VSG gene family.

Two pathways of homologous recombination in Trypanosoma brucei

African 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.

Antigenic variation in Trypanosoma brucei: joining the DOTs.

The survival ofTrypanosoma brucei relies on the sucessive expression of a single surface protein gene from a family of around 1,000 genes. This switching appears to be partly dictated by epigenetic changes in chromatin.

Distinct roles for two RAD51-related genes in Trypanosoma brucei antigenic variation

In Trypanosoma brucei, DNA recombination is crucial in antigenic variation, a strategy for evading the mammalian host immune system found in a wide variety of pathogens. T.brucei has the capacity to encode >1000 antigenically distinct variant surface glycoproteins (VSGs). By ensuring that only one VSG is expressed on the cell surface at one time, and by periodically switching the VSG gene that is expressed, T.brucei can evade immune killing for prolonged periods. Much of VSG switching appears to rely on a widely conserved DNA repair pathway called homologous recombination, driven by RAD51. Here, we demonstrate that T.brucei encodes a further five RAD51-related proteins, more than has been identified in other single-celled eukaryotes to date. We have investigated the roles of two of the RAD51-related proteins in T.brucei, and show that they contribute to DNA repair, homologous recombination and RAD51 function in the cell. Surprisingly, however, only one of the two proteins contributes to VSG switching, suggesting that the family of diverged RAD51 proteins present in T.brucei have assumed specialized functions in homologous recombination, analogous to related proteins in metazoan eukaryotes.