Interaction of human telomerase with its primer substrate.
Telomerase is a ribonucleoprotein responsible for maintaining the ends of linear chromosomes in nearly all eukaryotic cells. In humans, expression of the enzyme is limited primarily to the germ line and progenitor cell populations. In the absence of telomerase activity, telomeres shorten with each cell division until a critical length is reached, which can result in the cessation of cell division. The enzyme is required for cell immortality, and its activity has been detected in the vast majority of human tumors. Because of this, telomerase is an attractive target for inhibition in anticancer therapy. To learn more about the biochemistry of the human enzyme and its substrate recognition, we have examined the binding properties of single-stranded oligonucleotide primers that serve as telomerase substrates in vitro. We have used highly purified human enzyme and employed a two-primer method for determining the dissociation rates of these primers. Primers having sequence permutations of (TTAGGG)(3) were found to have considerably different affinities. They had t(1/2) values that ranged from 14 min to greater than 1200 min at room temperature. A primer ending in the GGG register formed the most stable complex with the enzyme. This particular register imparted stability to a nontelomeric primer resulting in a nearly 100-fold decrease in the k(off). We have found that interactions of telomerase with primer substrates are stabilized mainly by contacts with the protein subunit of the enzyme (hTERT). Base-pairing between the primer and the template region of telomerase contributes minimally to its stabilization.
Wallweber
Gerald
G
Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025, USA.
Gryaznov
Sergei
S
Pongracz
Krisztina
K
Pruzan
Ronald
R
D001667:Binding, Competitive; D020134:Catalytic Domain; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007700:Kinetics; D009154:Mutation; D012313:RNA / Q000737:chemistry; D013379:Substrate Specificity; D019098:Telomerase / Q000737:chemistry* / Q000302:isolation & purification; D013698:Templates, Genetic
D017931:DNA Primers; D006151:Guanosine; D012313:RNA; D019098:Telomerase