STRINGENT SEQUENCE REQUIREMENTS FOR THE FORMATION OF HUMAN TELOMERES

In human cells, transfection of telomeric T(2)AG(3) repeats induces the formation of functional telomeres at previously interstitial sites. We report that telomere formation has stringent sequence requirements, While (T(2)AG(3))(n) telomere seeds formed telomeres in approximate to 70% of the transfected cells, five T(2)AG(3)-related heterologous telomeric DNAs seeded new telomeres in <5% of the transfectants. Telomere formation did not correlate with the ability of human telomerase to elongate telomeric sequences in vitro. Homologous recombination is probably also not involved because a (T(2)AG(3))(n) telomere seed with nontelomeric DNA at 160-bp intervals formed new telomeres frequently. Instead, the sequence dependence of telomere formation matched the in vitro binding requirements for the mammalian T(2)AG(3) repeat binding factor (TRF). Human TRF failed to bind ineffective heterologous telomere seeds and had a 4-fold lower affinity for (T(2)AG(5))(2)T(2)AG(3) repeats, which seeded telomeres with reduced frequency. The results suggest that telomere seeds interact with TRF and predict that mammalian artificial chromosomes will require wild-type telomeric repeats at, or near, their termini.