T-loop assembly in vitro involves binding of TRF2 near the 3 ' telomeric overhang

Mammalian telomeres contain a duplex TTAGGG-repeat tract terminating in a 3 ' single-stranded overhang. TRF2 protein has been implicated in remodeling telomeres into duplex lariats, termed t-loops, in vitro and t-loops have be en isolated from cells in vivo. To examine the features of the telomeric DN A essential for TRF2-promoted looping, model templates containing a 500 bp double-stranded TTAGGG tract and ending in different single-stranded overha ngs were constructed. As assayed by electron microscope, looped molecules c ontaining most of the telomeric tract are observed with TRF2 at the loop ju nction. A TTAGGG-3' overhang of at least six nucleotides is required for lo op formation. Termini with 5' overhangs, blunt ends or 3' termini with non- telomeric sequences at the junction are deficient in loop formation. Additi on of non-telomeric sequences to the distal portion of a 3' overhang beginn ing with TTAGGG repeats only modestly diminishes looping. TRF2 preferential ly localizes to the junction between the duplex repeats and the single-stra nded overhang. Based on these findings we suggest a model for the mechanism by which TRF2 remodels telomeres into t-loops.