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Title: CAG.CTG trinucleotide repeat instability in the E.coli chromosome
Author: Zahra, Rabaab
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
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In order to identify the molecular basis of genetic instability, a polymerization-independent strategy is developed to generate expanded repeat arrays. The repeat tracts are integrated in the 5’end of lacZ gene in the Escherichia coli chromosome. Using this model system, instability is studied in wild type E. coli 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 II (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. coli 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. 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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available