In vitro and in vivo reconstitution and stability of vertebrate chromosome ends

Abstract

Telomeres are essential repetitive sequences at the ends of chromosomes that prevent chromosome fusion and degradation. We have successfully recapitulated these two protective functions in vivo and in vitro by incubating blunt-end DNA constructs having vertebrate telomeric ends in Xenopus eggs and egg extracts. Constructs with telomeric ends are stable as linear molecules; constructs with non-telomeric ends undergo intramolecular fusion. In extracts, 99.8% of the telomeric constructs from 78 to 700 bp in length are assembled into 'model telomeres' in < 5 min and have an extrapolated halflife of > 3.5 years. Non-telomeric constructs circularize with first order kinetics and a half-life of 4 h. In living eggs the telomeric constructs are protected from fusion and degradation. The stability of the telomeric constructs is not due to covalent processing. Extract can protect ~ 100 pM telomeric ends (equivalent to 1.7 x 107 ends/egg) even in the presence of a 20-fold excess of double-stranded telomeric DNA, suggesting that protection requires end-specific factors. Constructs with (TTGGGG), repeats are unstable, suggesting that short tracts of this and other telomere-like sequences found within human telomeres could lead to genome instability if exposed by partial telomere erosion during aging.