Fidelity of primate cell repair of a double-strand break within a (CTG).(CAG) tract. Effect of slipped DNA structures.
At least 15 human diseases are caused by the instability of gene-specific (CTG).(CAG) repeats. The precise mechanism of instability remains unknown, though bacterial and yeast models have suggested a role for aberrant repair of double-strand breaks (DSBs). Using an established primate DSB repair system, we have investigated the fidelity of repair of a DSB within a (CTG).(CAG) repeat tract. DSB repair substrates were generated from plasmids that are stably replicated in their circular form, permitting us to highlight the effects of DSB repair on repeat stability and minimize the contribution of replication. DSBs were introduced into repeat-containing plasmids using a unique BsmI site, such that the entire repeat tract comprised one free end of the linearized plasmid. Substrates containing 17, 47, and 79 repeats, in either their linear duplex form or containing slipped structures (out-of-register interstrand mispairings at repeat sequences), were transiently transfected into primate cells. Linearized plasmids with repeats were repaired with mildly reduced efficiency, while the presence of slipped structures considerably reduced repair efficiency. The repaired products were characterized for alterations within the repeat tract and flanking sequence. DSB repair induced predominantly repeat deletions. Notably, a polarized/directional deletion effect was observed, in that the repetitive end of the DSB was preferentially removed. This phenomenon was dramatically enhanced when slipped structures were present within the repeat tract, providing the first evidence for error-prone processing of slipped-strand structures. These results suggest the existence of primate nuclease activities that are specific for (CTG).(CAG) repeats and the structures they form.
Marcadier
Julien L
JL
Department of Molecular & Medical Genetics, University of Toronto, Ontario M5A 1X8, Canada.
Pearson
Christopher E
CE
D000818:Animals; D019556:COS Cells; D004249:DNA Damage; D004260:DNA Repair; D017353:Gene Deletion; D006801:Humans; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D014162:Transfection; D019680:Trinucleotide Repeat Expansion