During its life cycle, Trypanosoma brucei shuttles between a mammalian host and the tsetse fly vector. In the mammalian host, immune evasion of T. brucei bloodstream form (BSF) cells relies on antigenic variation, which includes monoallelic expression and periodic switching of variant surface glycoprotein (VSG) genes. The active VSG is transcribed from only 1 of the 15 subtelomeric expression sites (ESs). During differentiation from BSF to the insect-resident procyclic form (PCF), the active ES is transcriptionally silenced. We used mass spectrometry-based interactomics to determine the composition of telomere protein complexes in T. brucei BSF and PCF stages to learn more about the structure and functions of telomeres in trypanosomes. Our data suggest a different telomere complex composition in the two forms of the parasite. One of the novel telomere-associated proteins, TelAP1, forms a complex with telomeric proteins TbTRF, TbRAP1 and TbTIF2 and influences ES silencing kinetics during developmental differentiation.

Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to offspring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation.

The fluidity of Trypanosoma brucei ′s dense coat of GPI-anchored variant surface glycoproteins (VSGs) is fundamental for the survival of the parasite. In order to maintain the integrity of the coat, it is recycled on the time scale of a few minutes. This is surprisingly fast as endo- and exocytosis take place in the same small membrane invagination called the flagellar pocket. Here, we present measurements of VSG dynamics on the single-molecule level in living trypanosomes. A large number of short protein trajectories sampling the parasite's surface were analysed in two distinct scenarios: diffusion and directed motion. To this end, we employed a previously published algorithm and implemented two extensions to consider rim effects as well as localisations errors inherent to single-mole tracking. Neglect of the latter can have a significant distortive effect on the measured diffusion coefficient; in our case resulting in an underestimation by 20%. We found large heterogeneity in the local diffusion coefficients and velocities with a surprisingly high average value of D = 1.00 μm 2/s and v = 1.99 μm/s, respectively. To decide on the locally dominant motion mode, we present a guideline based on random walk simulations. We find that VSG dynamics is indeed dominated by diffusion. Complementary simulations on long time scales not accessible in the experiment showed that passive VSG randomisation is fast enough to prevent re-endocytosis newly exocytosed VSGs and to accomplish turnover of the full VSG coat within a few minutes.

Richard McCulloch: phone +44 (0) 1413305946; email: Richard.McCulloch@glasgow.ac.uk # These authors contributed equally to this work. AbstractMaintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to offspring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage, often at the cost of mutation. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to hostparasite interactions. Here, we describe a dual function of translesion PolN in African trypanosomes. Previously we demonstrated that PolN is associated with telomeric sequences and now we show that RNAi-mediated depletion of PolN results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. Depletion of PolN leads to chromosome segregation defects and accumulation of DNA damage. We also show that PolN displays discrete localisation at the nuclear periphery in the absence of exogenous DNA damage. In addition, we demonstrate that PolN depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this translesion DNA polymerase to host immune evasion by antigenic variation. Introduction Accurate duplication of the genome is a critical component of the cell cycle of all organisms. Two pathways contribute to accurate genome duplication: copying of the genome, and repair of DNA damage. Eukaryotic cells encode a wide range of DNA polymerases (Pols) that are required for DNA synthesis, allowing genome duplication, and for repair of DNA damage (review in [1]). Eukaryotic DNA Pols are divided into four different families (A, B, X and Y) based on sequence and structural homologies. Nuclear DNA Pols that direct the accurate copying of the genome belong to the B family, while mitochondrial genome replication is catalysed by an A family DNA Pol [2]. DNA Pols that act in DNA repair span all families, as do so-called translesion DNA Pols, which straddle DNA repair and replication activities because their activity is required whenever replicative DNA Pols encounter lesions in the template strand that must be bypassed to allow genome duplication [3];[4];[5]. In general,DNA replication is a high fidelity process with an extremely low error rate [6]. This is due to a combination of the ability of replicative DNA Pols to efficiently select the correct nucleotide to incorporate into the newly synthesized DNA strand and proofreading activity of the Pols, which permits the excision of occasionally incorrectly inserted nucleotides. Additionally, postreplicative repair mechanisms further reduce overall error rates by removing mispaired or damaged bases [7]. Although the wide range of DNA repair mechanisms available to all cells can efficiently detect and rem...