Ever shorter telomere 1 (EST1)-dependent reverse transcription by Candida telomerase in vitro: evidence in support of an activating function.
Telomerase is an RNA-protein complex responsible for the extension of one strand of the telomere terminal repeats. Analysis of the telomerase complex in the budding yeast Saccharomyces cerevisiae has revealed the presence of one catalytic protein subunit (Est2p/TERT) and at least two noncatalytic components (Est1p and Est3p). The TERT subunit is essential for telomerase function, both in vitro and in vivo. In contrast, the Est1p and Est3p subunits, although required for telomere extension in vivo, have not been shown to affect enzyme activity in vitro. We recently identified orthologues of the Saccharomyces telomerase subunits in Candida albicans (named CaTERT, CaEst1p, and CaEst3p). Analysis of telomerase from the Candida Caest1-Delta strains revealed a primer-specific defect in its activity in vitro: The mutant enzyme was impaired in its ability to extend some, but not all, telomeric primers. The CaEst1p-responsive primers have 3' ends that are clustered in two loci within the 23-bp Candida telomere repeat. The degree of CaEst1p-dependence was modulated by the length and sequence of the 5' ends. For CaEst1p-dependent primers, the wild-type enzyme consistently exhibited a greater V(max) than the mutant enzyme in kinetic studies. These results suggest that CaEst1p augments the ability of telomerase to reverse-transcribe through selected barriers in the telomere repeat by acting as an allosteric activator and provide the basis for a functional in vitro assay for a noncatalytic protein component of the telomerase complex.
Singh
Sunitha M
SM
Department of Microbiology and Immunology, W. R. Hearst Microbiology Research Center, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.
Lue
Neal F
NF
D001483:Base Sequence; D002175:Candida / Q000201:enzymology*; D017931:DNA Primers; D007700:Kinetics; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019098:Telomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D017931:DNA Primers; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; C099024:EST1 protein, S cerevisiae; D012194:RNA-Directed DNA Polymerase; D019098:Telomerase