In addition, competition assays showed that this complex was unaffected by excess of poly [dI-dC] [dI-dC] (Fig 6, lane 3), used as the non-specific competitor, but it was almost completely abolished in the presence of excess unlabeled LaTEL (Fig 6, lane 4). Supershift experiments RG-7388 purchase using anti-LaTRF serum were done in the presence of competitor to confirm that LaTRF was actually involved in the formation of the retarded band (Fig 6, lane 5). Note that the retarded shifted band disappeared due to the competition by non-labeled LaTEL. Thus, these results indicate

that LaTRF is in part responsible for the binding activity shown in these extracts and is probably a component of the Leishmania telomeric complex. Chromatin immunoprecipitation experiments also suggested that LaTRF is a telomeric protein. The anti-LaTRF serum immunoprecipitated

L. amazonensis telomeric DNA (Tel1) in vivo (Fig 7 – left) but did not immunoprecipitate the GT-rich kinetoplast DNA (kDNA) (Fig 7 – right). The kDNA control represented by the UMS (universal mini-circle sequence) albeit GT-rich, is very representative of the general base composition of Leishmania genomic DNA. In addition, it is a good control, since we were able to show that it was co-immunoprecipitated by two other Leishmania telomeric protein [17, 23]. In a previous study, we described LaTBP1, a protein that specifically binds telomeric and GT-rich DNA in MK5108 Leishmania. LaTBP1 has a centrally positioned Myb-like DNA binding domain and is most likely a non-telobox protein that is apparently related to the multifunctional yeast RAP1 telomeric protein

and TFIIIB B”" transcription factor [17]. Together with the putative LaTRF described here, these are the only descriptions of proteins bearing a Myb-like DNA binding domain that interact with double-stranded telomeric DNA in Leishmania. Figure Endonuclease 7 LaTRF interacts with telomeric DNA in vivo. Chromatin immunoprecipitation (CHIP) of mid-log phase promastigotes cells using anti-LaTRF. Control experiments were done with chromatin immunoprecipitated in the presence of pre-immune serum and without serum (mock). Total DNA (input) corresponds to 10% and 1% of the amount of DNA in 108 cells cross-linked with the chromatin. Slot-blots were hybridized with 5′ end-labeled Tel1 probe (left) and re-hybridized with the kDNA probe (right). As mentioned here and elsewhere [26], the huge evolutionary distance between this protozoan and higher eukaryotes presents a barrier when searching for protein homologues in the genomes of these parasites. For example, no TRF1 homologues were found in trypanosomatid genomes but the expression of hTRF1 in procyclic forms of T. brucei caused telomere shortening and cell cycle arrest, probably by displacing an unknown endogenous telomeric factor [29].