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 been isolated from cells in vivo. To examine the features of the telomeric DNA essential for TRF2-promoted looping, model templates containing a 500 bp double-stranded TTAGGG tract and ending in different single-stranded overhangs were constructed. As assayed by electron microscopy, looped molecules containing most of the telomeric tract are observed with TRF2 at the loop junction. A TTAGGG-3' overhang of at least six nucleotides is required for loop formation. Termini with 5' overhangs, blunt ends or 3' termini with non-telomeric sequences at the junction are deficient in loop formation. Addition of non-telomeric sequences to the distal portion of a 3' overhang beginning with TTAGGG repeats only modestly diminishes looping. TRF2 preferentially localizes to the junction between the duplex repeats and the single-stranded overhang. Based on these findings we suggest a model for the mechanism by which TRF2 remodels telomeres into t-loops.
Stansel
R M
RM
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295, USA.
de Lange
T
T
Griffith
J D
JD
D004277:DNA, Single-Stranded / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000648:ultrastructure; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid; D016615:Telomere / Q000378:metabolism* / Q000648:ultrastructure*; D035341:Telomeric Repeat Binding Protein 2
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D035341:Telomeric Repeat Binding Protein 2