Double-strand DNA breaks (DSBs) occur during normal cell metabolism and are lethal unless repaired. <i>E. coli </i>repairs DSBs using a pathway that involves homologous recombination. The mechanisms involved in this process were investigated by manipulating the <i>Eco</i>KI restriction-modification system of <i>E. coli</i> 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 <i>recG </i>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/<i>dif </i>system that is involved in chromosome dimer resolution. The deleterious effect of <i>xerC</i> mutations on <i>recG </i>and <i>ruvAC </i>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 <i>yqgF </i>gene is an alternative HJ resolvase was investigated. <i>yqgF </i>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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:663481 |
| Date | January 2007 |
| Creators | Wardrope, Laura |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13208 |
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