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Title: Repair of double-strand DNA breaks in Escherichia coli
Author: Wardrope, Laura
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2007
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Double-strand DNA breaks (DSBs) occur during normal cell metabolism and are lethal unless repaired. E. coli repairs DSBs using a pathway that involves homologous recombination. The mechanisms involved in this process were investigated by manipulating the EcoKI restriction-modification system of E. coli so that the restriction activity cleaves chromosomes to produce DSBs. The viability of recombination and repair mutants was measured following the induction of DSBs. The results show that RecG and RuvABC facilitate the survival of DSBs. Surprisingly, RuvABC was able to promote survival even when recombination could not be initiated. Pulsed field gel electrophoresis (PFGE) was carried out on the genomic DNA of mutants exposed to DSBs. This allowed Holliday junctions (HJs) linking the chromosomes of strains lacking RuvABC to be detected. Most significantly, the PFGE phenotype of a recG mutant mirrored that of the wild-type, suggesting that the RecG protein is not involved in the resolution of HJs. The outcome of HJ resolution to form crossover or non-crossover products was also investigated in mutants exposed to DSBs by measuring the effect on viability of inactivating the XerCD/dif system that is involved in chromosome dimer resolution. The deleterious effect of xerC mutations on recG and ruvAC mutants was approximately 10-fold greater than on wild-type. These results prompted an interesting discussion as to how the functions of the products of these genes interact in the cell. Finally, the theory that the product of the essential yqgF gene is an alternative HJ resolvase was investigated. yqgF was placed under the control of an inducible promoter and the effect of depleting YqgF levels on survival of DSBs was measured. No evidence to suggest that YqgF can resolve HJs was found.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available