Purification of telomerase from Euplotes aediculatus: requirement of a primer 3' overhang.
Telomerase is a ribonucleoprotein enzyme that uses its internal RNA moiety as a template for synthesis of telomeric repeats at chromosome ends. Here we report the purification of telomerase from Euplotes aediculatus by affinity chromatography with antisense 2'-O-methyl oligonucleotides, a method that was developed for small nuclear ribonucleoprotein particles (snRNPs). Elution of bound ribonucleoprotein from the antisense oligonucleotide under nondenaturing conditions was achieved by a novel approach, using a displacement oligonucleotide. Polypeptides of 120 kDa and 43 kDa (a doublet) copurify with the active telomerase and appear stoichiometric with telomerase RNA. A simple model for DNA end replication predicts that after semiconservative DNA replication, telomerase will extend the newly synthesized, blunt-ended leading strand. We show that purified Euplotes telomerase has no activity with blunt-ended primers. Instead, efficient extension requires 4 to 6 single-stranded nucleotides at the 3' end. Therefore, this model predicts the existence of other activities such as helicases or nucleases that generate a single-stranded 3' end from a blunt end, thus activating the end for telomerase extension.
Lingner
J
J
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado 80309-0215, USA.
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D000818:Animals; D002846:Chromatography, Affinity; D004277:DNA, Single-Stranded / Q000378:metabolism; D016812:Euplotes / Q000201:enzymology*; D006860:Hydrogen Bonding; D009690:Nucleic Acid Conformation; D012261:Ribonucleoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D019098:Telomerase / Q000302:isolation & purification* / Q000378:metabolism
D004277:DNA, Single-Stranded; D012261:Ribonucleoproteins; D019098:Telomerase