Dere, Ruhee J. (2006-08). The molecular mechanisms involved in the genetic instability of the CCTG. CAGG repeats associated with myotonic dystrophy type 2. Doctoral Dissertation.
Thesis
Myotonic dystrophy type 2 (DM2) is caused by the extreme expansion (from < 30 repeats in normal individuals to ~ 11,000 for the full mutation in certain patients) of the repeating tetranucleotide CCTGA?CAGG sequence in the intron of the zinc finger protein 9 (ZNF9) gene. The genetic instabilities of the CCTGA?CAGG repeats were investigated to evaluate the molecular mechanisms responsible for these massive expansions. The effects of replication, recombination, repair and transcription on the genetic instabilities have been investigated in COS-7 cells and E. coli model systems. A replication assay was established in COS-7 cells wherein the CCTGA?CAGG repeats cloned proximal to the SV40 origin of replication resulted in expansions and deletions in a length and orientation-specific manner, whereas the repeats cloned distal to the same origin were comparatively stable. These results fit with our data obtained from biochemical studies on synthetic oligonucleotides since these biochemical studies revealed that the d(CAGG)26 oligomer had a marked propensity to adopt a hairpin structure as opposed to its complementary d(CCTG)26 that lacked this capacity. Furthermore, a genetic assay in E. coli was used to monitor the intramolecular frequency of recombination. This assay revealed that the tetranucleotide repeats were indeed hot spots for recombination. Moreover, studies conducted in SOS-repair mutants showed that recombination frequencies were much lower in a SOSA? strain as compared to a SOS+ strain. However, experiments conducted to ascertain the level of induction of the SOS response revealed that the SOS pathway was not stimulated in our studies. These results revealed that although breaks may occur within the repeats, the damage is most likely repaired without induction of the SOS response contrary to previous beliefs. Thus, a complex interplay of replication, recombination, and repair is likely responsible for the expansions observed in DM2.
