A Trypanosoma brucei Protein Complex That Binds G-overhangs and Co-purifies with Telomerase Activity

• Published in: The Journal of biological chemistry Vol. 277; no. 2; pp. 896 - 906
• Main Authors: Cano, Maria Isabel N, Blake, Julie Johnson, Blackburn, Elizabeth H, Agabian, Nina
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 11.01.2002
• Subjects: Animals; Cell Fractionation; complex C3; DNA - metabolism; DNA-Binding Proteins - metabolism; Humans; Macromolecular Substances; Oligonucleotides - metabolism; Protozoan Proteins - chemistry; Protozoan Proteins - isolation & purification; Protozoan Proteins - metabolism; ribonuclease A; RNA - metabolism; RNase A; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Telomerase - chemistry; Telomerase - isolation & purification; Telomerase - metabolism; Trypanosoma brucei; Trypanosoma brucei brucei - chemistry; Trypanosoma brucei brucei - metabolism; Ultraviolet Rays
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M104111200

The chromosomal ends of Trypanosoma brucei , like those of most eukaryotes, contain conserved 5′-TTAGGG-3′ repeated sequences and are maintained by the action of telomerase. Fractionated T. brucei cell extracts with telomerase activity were used as a source of potential regulatory factors or telomerase-associated components that might interact with T. brucei telomeres. Electrophoretic mobility shift assays and UV cross-linking were used to detect possible single-stranded telomeric protein·DNA complexes and to estimate the approximate size of the protein constituents. Three single-stranded telomeric protein·DNA complexes were observed. Complex C3 was highly specific for the G-strand telomeric repeat sequence and shares biochemical characteristics with G-rich, single-stranded telomeric binding proteins and with components of the telomerase holoenzyme described in yeast, ciliates, and humans. Susceptibility to RNase A or chemical nuclease (hydroxyl radical) pre-treatment showed that complex C3 was tightly associated with an RNA component. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry was used to estimate the molecular mass of the peptides obtained by in-gel Lys-C digestion of low abundance C3-associated proteins. The molecular masses of the peptides showed no homologies with other proteins from trypanosomes or with any protein in the data bases screened.