16 research outputs found
Study for activation of human telomerase reverse transcriptase, as major component of cancer, expressed in E. coli
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N-terminal Domain of Yeast Telomerase Reverse Transcriptase: Recruitment of Est3p to the Telomerase Complex
Telomerase is a reverse transcriptase that maintains chromosome ends. The N-terminal half of the catalytic protein subunit (TERT) contains three functional domains (I, II, and III) that are conserved among TERTs but not found in other reverse transcriptases. Guided by an amino acid sequence alignment of nine TERT proteins, mutations were introduced into yeast TERT (Est2p). In support of the proposed alignment, mutation of virtually all conserved residues resulted in loss-of-function or temperature sensitivity, accompanied by telomere shortening. Overexpression of telomerase component Est3p led to allele-specific suppression of the temperature-sensitive mutations in region I, suggesting that Est3p interacts with this protein domain. As predicted by the genetic results, a lethal mutation in region I resulted in loss of Est3p from the telomerase complex. We conclude that Est2p region I is required for the recruitment of Est3p to yeast telomerase. Given the phylogenetic conservation of region I of TERT, this protein domain may provide the equivalent function in all telomerases
Exo1 Roles for Repair of DNA Double-Strand Breaks and Meiotic Crossing Over in Saccharomyces cerevisiae
The Product of the Survival of Motor Neuron (SMN) Gene is a Human Telomerase-associated Protein
Telomerase is a ribonucleoprotein (RNP) complex that is minimally composed of a protein catalytic subunit, the telomerase reverse transcriptase (TERT), and an RNA component, the telomerase RNA. The survival of motor neuron (SMN) gene codes for a protein involved in the biogenesis of certain RNPs. Here, we report that SMN is a telomerase-associated protein. Using in vitro binding assays and immunoprecipitation experiments, we demonstrate an association between SMN and the telomerase RNP in vitro and in human cells. The specific immunopurification of SMN from human 293 cells copurified telomerase activity, suggesting that SMN associates with a subset of the functional telomerase holoenzyme. Our results also indicate that the human telomerase RNA and the human (h) TERT are not associated with Sm proteins, in contrast to Saccharomyces cerevisiae telomerase. Immunofluorescence analysis showed that hTERT does not specifically colocalize with wild-type SMN in gems or Cajal bodies. However, a dominant-negative mutant of SMN (SMNΔN27) previously characterized to elicit the cellular reorganization of small nuclear RNPs caused the accumulation of hTERT in specific SMNΔN27-induced cellular bodies. Furthermore, coexpression of SMNΔN27 and hTERT in rabbit reticulocyte lysates decreased the efficiency of human telomerase reconstitution in vitro. Our results establish SMN as a novel telomerase-associated protein that is likely to function in human telomerase biogenesis