EFFECT OF DNA SECONDARY STRUCTURE ON HUMAN TELOMERASE ACTIVITY

Telomeres are specialized DNA-protein complexes located at the chromos ome ends. The guanine-rich telomeric sequences have the ability to for m G-quadruplex structures under physiological ionic conditions in vitr o. Human telomeres are maintained through addition of TTAGGG repents b y the enzyme telomerase. To determine a correlation between DNA second ary structure and human telomerase, telomerase activity in the presenc e of various metal cations was monitored. Telomerase synthesized a lar ger proportion of products corresponding to four, five, eight, and nin e full repeats of TTAGGG in 100 mM K+ and to a lesser extent in 100 mM Na+ when a d(TTAGGG)(3) input primer was used. A more even product di stribution was observed when the reaction mixture contained no added N a+ or K+. Increasing concentrations of Csi resulted in a loss of proce ssivity but not in the distinct manner observed in K+. When the input primer contained 7-deaza-dG, the product distribution resembled that o f reactions without K+ even in the presence of 100 mM K+. Native polya crylamide gel electrophoresis indicated that d(TTAGGG)(4), d(TTAGGG)(5 ), d(TTAGGG)(8), and d(TTAGGG)(9) formed compact structures in the pre sence of K+. The oligonucleotide d(TTAGGG)(4) had a UV spectrum charac teristic of that of the G-quadruplex only in the presence of K+ and Na +. A reasonable explanation for these results is that four, five, eigh t, and nine repeats of TTAGGG form DNA secondary structures which prom ote dissociation of the primer from telomerase. This suggests that tel omerase activity in cells can be modulated by the secondary structure of the DNA template. These findings are of probable relevance to the c oncept of telomerase as a therapeutic target for drug design.