Cag.,ctg Trinucleotide Repeat Instability in the E. Coli Chromosome

Expanded CAG-CTG trinucleotide repeat tracts are associated with a number of hereditary neurodegenerative and neuromuscular diseases such as Huntington's disease, myotonic dystrophy and spinocerebellar ataxias. These diseases are characterized by the phenomenon of genetic anticipation, which is defined by a decrease in the age of onset and an increase in severity of the disease with successive generations. The mutational mechanism of repeat instability is still not fully understood. In order to identify the molecular basis of genetic instability, a polymerizationindependent strategy is developed to generate expanded repeat arrays. The repeat tracts are integrated in the 5'end of lacZ gene in the Escherichia co/i chromosome. Using this model system, instability is studied in wild type E. co/i and in strains deficient in cellular pathways such as DNA repair, replication and recombination. The work demonstrates that instability (expansion and contraction) in wild type cells is length and orientation dependent. Longer tracts are more unstable than shorter ones and the orientation where CAG repeats are on the leading strand template is more unstable than the opposite where CTG repeats are on the leading strand template. This orientation-dependence of CAG-CTG trinucleotide repeat instability is determined by the proofreading subunit of DNA polymerase III (DnaQ) in the presence of the hairpin nuclease SbcCD. The analysis of the sizes of deletions observed in wild type and mutant cells is consistent with the formation of secondary structures in vivo. The mismatch repair pathway does not affect the instability of CTG repeats in the E. co/i chromosome but influences the CAG orientation. It is suggested that MutS stabilizes CAG repeats by initiating a "repair" process and protecting hairpins from SbcCD, which can cleave hairpins in the presence of MutL and MutH. No effect of recombination genes is observed on repeat instability in the E. coli chromosome. No effect of transcription is observed in the wild type or mutant strains tested in this work. A mutation in mfd gene also does not affect instability. Furthermore, CAGCTG repeats influence the yield of f3-galactosidase in an orientation dependent manner. Finally, the roles of two helicases, Rep and UvrD are analyzed. A mutation in rep helicase strongly destabilizes CTG repeats with no effect on the CAG orientation. UvrD mutants show instability in both orientations. The increase in instability in the uvrD mutant depends on RecF in the CTG orientation.