The chromosomal ends of Leishmania (Leishmania) amazonensis contain conserved 5¢-TTAGGG-3¢ telomeric repeats. Protein complexes that associate in vitro with these DNA sequences, Leishmania amazonensis G-strand telomeric protein (LaGT1-3), were identified and characterized by electrophoretic mobility shift assays and UV cross-linking using protein fractions purified from S100 and nuclear extracts. The three complexes did not form (a) with doublestranded DNA and the C-rich telomeric strand, (b) in competition assays using specific telomeric DNA oligonucleotides, or (c) after pretreatment with proteinase K. LaGT1 was the most specific and did not bind a Tetrahymena telomeric sequence. All three LaGTs associated with an RNA sequence cognate to the telomeric G-rich strand and a complex similar to LaGT1 is formed with a double-stranded DNA bearing a 3¢ G-overhang tail. The protein components of LaGT2 and LaGT3 were purified by affinity chromatography and identified, after renaturation, as 35 and 52 kDa bands, respectively. The £ 15 kDa protein component of LaGT1 was gel-purified as a UV cross-linked complex of 18-20 kDa. Peptides generated from trypsin digestion of the affinity and gel-purified protein bands were analysed by matrix-assisted laser desorption/ ionization-time of flight and electrospray ionization tandem mass spectrometry. The fingerprint and amino acid sequence analysis showed that the protein components of LaGT2 and of LaGT3 were, respectively, similar to the kinetoplastid Rbp38p and to the putative subunit 1 of replication protein A of Leishmania spp., whereas the £ 15 kDa protein component of LaGT1 was probably a novel Leishmania protein.Keywords: affinity purification; EMSA; Leishmania amazonensis; mass spectrometry; telomeric proteins.In almost all eukaryotes, including the pathogenic protozoan Leishmania (Leishmania) amazonensis, telomeres are nucleoprotein complexes formed by tandem repeats of conserved DNA sequences associated with proteins [1,2]. One of the telomere strands is G-rich and runs 5¢ fi 3¢ towards the end of the chromosomes, where it forms a single-stranded protrusion or 3¢ G-overhang [3]. The G-rich strand is the substrate for telomerase and for other telomere binding proteins involved in telomere length regulation and maintenance [4,5]. The length of this G-rich telomere extension appears to be cell cycle regulated in humans and yeast [6][7][8] and its loss leads to genome instability and chromosomal end fusion through the activation of DNA damage checkpoints [5,9].Proteins associated with both double-stranded and G-rich single-stranded telomeric DNA and with accessory proteins have been described in many eukaryotes. These proteins form a high order nucleoprotein complex that functions mainly to maintain the genome stability by regulating telomerase activity, the expression of genes positioned at telomeres, and the capping of chromosome ends to protect them from degradation and fusions [10,11]. For example, during the S phase, which is the period of increased single-strand extensi...

Telomeres are protein-DNA complexes that protect chromosome ends from degradation and fusion. In Leishmania spp., telomeric DNA comprises a conserved TTAGGG repeat and is maintained by telomerase. Telomerase is a multisubunit enzymatic complex that ensures the complete DNA replication by adding new telomeric repeats to the G-rich strand. In this report we aimed to purify and study the biochemical properties of Leishmania amazonensis telomerase. In a first trial we used affinity chromatography with antisense 2'-O-methyl oligonucleotide without success since the Leishmania telomerase, similarly to Trypanosoma cruzi enzyme, was not eluted by competition, but instead, it remained bound to the column. Partially purified L. amazonensis telomerase activity was achieved by fractionation of extracts on complementary ion exchange and Heparin columns. Further purification of these fractions on a G-rich telomeric DNA affinity chromatography enriched for telomerase activity. The knowledge of telomerase characteristics in Leishmania could help to develop new strategies to overcome leishmaniasis.

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