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Title: Expanded CTG trinucleotide repeats stimulate homologous recombination in Escherichia coli
Author: Blackwood, John Kenneth
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
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Expanded trinucleotide repeats (TNRs) (e.g. CAG, CTG, CCG) cause 40 different human diseases, however the molecular mechanism underlying the expansion of TNRs is poorly understood. This work describes the integration, in the chromosome of the bacterium Escherichia coli of differently sized CAG and CTG TNRs into the start of the lacZ gene and of a zeocin resistance recombination reporter substrate into the nearby gene, cynX.  We show that TNRs stimulate recombination at cynX in a length dependent manner. Furthermore, stimulation of recombination is dependent on TNR orientation with respect to the origin of replication. Experiments indicate that zeocin recombination is reduced in E. coli mutants for double strand break repair (recA and recB); but not for gap repair (recR). TNR induced stimulation of recombination is shown to be independent of the DNA hairpin nuclease SbcCD arguing against any role of secondary structure formed by TNRs. The formation of DSBs by TNRs is investigated using pulse field gel electrophoresis (PFGE). Since reversed replication forks can initiate homologous recombination (HR), this thesis investigates the possibility that TNRs lead to replication form reversal (RFR). We test this hypothesis by assaying for HR using zeocin recombination reporter substrates positioned proximal and distal with respect to the origin of replication. We show that TNR induced HR is lower at the distal site. Furthermore, the protein UvrD is known to be essential for RFR in certain replication mutants. TNR dependent stimulation of HR is lost in uvrD mutants. Both these data support the hypothesis that TNRs can cause RFR.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available