SbcCD has been shown to cleave a DNA hairpin formed by a palindromic DNA sequence on the lagging strand template of the E. coli chromosome. This activity was exploited to create a unique system for inducing a single site-specific DNA double-strand break (DSB) once per replication cycle. First, this work shows that the SOS response induced by this DSB is only essential for viability following multiple cycles of cleavage and repair. Next, the SOS-inducible inhibitor of cell division SfiA is shown to be dispensable for survival, despite demonstrating that cleavage of the palindrome causes both an increase in cell size and a delay in nucleoid segregation. A model of the E. coli cell cycle is presented to reconcile the observation that growth under chronic DSB induced conditions has no effect on generation time despite causing an increase in cell size. This system of DSB induction was then coupled with fluorescence markers on both sides of the palindrome to visualise the consequence of the DSB in vivo. Cleavage of the DNA hairpin by SbcCD in a recAmutant was used to selectively degrade the chromosome that replicated the palindrome on the lagging strand of replication, allowing two genetically identical sister chromosomes to be distinguished. This approach was used to show that chromosome segregation in E. coli is not random, but results in the segregation of lagging strand replicated DNA to mid-cell and leading strand replicated DNA to cell poles. Finally, this system for visualising the site of an inducible DSB was optimised for use in various other mutant backgrounds to allow the events of DSB repair to be dissected. This work provides a solid basis for further investigation into the relationship between replication, recombination and chromosome segregation in the model organism E. coli.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:562844 |
| Date | January 2010 |
| Creators | White, Martin A. |
| Contributors | Leach, David |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/4088 |
Page generated in 0.0019 seconds