Abstract
Trypanosome antigenic variation, involving differential expression of variant surface glycoprotein (VSG) genes, has a strong association with telomeres and with DNA recombination. All expressed VSGs are telomeric, and differential activation involves recombination into the telomeric environment or silencing/activation of subtelomeric promoters. A number of pathogen contingency gene systems associated with immune evasion involve telomeric loci, which has prompted speculation that chromosome ends provide conditions conducive for the operation of rapid gene switching mechanisms. Ku is a protein associated with eukaryotic telomeres that is directly involved in DNA recombination and in gene silencing. We have tested the hypothesis that Ku in trypanosomes is centrally involved in differential VSG expression. We show, via the generation of null mutants, that trypanosome Ku is closely involved in telomere length maintenance, more so for a transcriptionally active than an inactive telomere, but exhibits no detectable influence on DNA double strand break repair. The absence of Ku and the consequent great shortening of telomeres had no detectable influence either on the rate of VSG switching or on the silencing of the telomeric promoters of the VSG subset that is expressed in the tsetse fly.
Highlights
The heterodimeric protein Ku, which consists of the subunits Ku70 and Ku80, associates tightly in a sequenceindependent fashion with free ends of double strand DNA and has been associated with a range of nuclear functions in different eukaryotes, including DNA repair, retrotransposition [4], gene silencing, transcriptional reinitiation [5], chromosome maintenance, and chromosome localization
Our analysis of telomere lengths revealed the telomere shortening phenotype that occurs in other eukaryotes, but the classical role in DNA damage repair by non-homologous end joining (NHEJ) was not detected
Our hypothesis that Ku is important in variant surface glycoprotein (VSG) recombinational or transcriptional control was not supported, revealing at least that the Kumediated telomere position effect (TPE) described for S. cerevisiae is not necessary for regulation of antigenic variation
Summary
There can be occasional transcriptional switches between BESs, the main route to VSG switching is the replacement of the VSG in the BES with a copy of a silent VSG [33], probably by homologous recombination [34]. It is not known yet whether special, telomere-associated mechanisms enhance recombination into BESs, and a role for telomereassociated recombination-promoting proteins is a possibility. We examine the functions of Ku in trypanosomes and test the hypothesis that it is important for VSG switching in the bloodstream stage and that it is important for the silencing of MVSG genes prior to the metacyclic stage
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