Transposon Tn7 preferentially inserts into GAA*TTC triplet repeats under conditions conducive to Y*R*Y triplex formation.
BACKGROUND
Expansion of an unstable GAA*TTC repeat in the first intron of the FXN gene causes Friedreich ataxia by reducing frataxin expression. Structure formation by the repeat has been implicated in both frataxin repression and GAA*TTC instability. The GAA*TTC sequence is capable of adopting multiple non-B DNA structures including Y*R*Y and R*R*Y triplexes. Lower pH promotes the formation of Y*R*Y triplexes by GAA*TTC. Here we used the bacterial transposon Tn7 as an in vitro tool to probe whether GAA*TTC repeats can attract a well-characterized recombinase.


RESULTS
Tn7 showed a pH-dependent preference for insertion into uninterrupted regions of a Friedreich ataxia patient-derived repeat, inserting 48, 39 and 14 percent of the time at pH 7, pH 8 and pH 9, respectively. Moreover, Tn7 also showed orientation and region specific insertion within the repeat at pH 7 and pH 8, but not at pH 9. In contrast, transposon Tn5 showed no strong preference for or against the repeat during in vitro transposition at any pH tested. Y*R*Y triplex formation was reduced in predictable ways by transposon interruption of the GAA*TTC repeat. However, transposon interruptions in the GAA*TTC repeats did not increase the in vitro transcription efficiency of the templates.


CONCLUSIONS
We have demonstrated that transposon Tn7 will recognize structures that form spontaneously in GAA*TTC repeats and insert in a specific orientation within the repeat. The conditions used for in vitro transposition span the physiologically relevant range suggesting that long GAA*TTC repeats can form triplex structures in vivo, attracting enzymes involved in DNA repair, recombination and chromatin modification.
Mancuso
Miriam
M
Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, Louisiana, United States of America.
Sammarco
Mimi C
MC
Grabczyk
Ed
E
D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004251:DNA Transposable Elements; D024202:Electrophoretic Mobility Shift Assay; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid
D004251:DNA Transposable Elements; D004247:DNA