The repair of DNA breaks in Escherichia coli and analysis of the bacterial recombination protein RecN

Wood, Stuart Robert (2009) The repair of DNA breaks in Escherichia coli and analysis of the bacterial recombination protein RecN. PhD thesis, University of Nottingham.

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Abstract

A DNA double-strand break is an exceptionally toxic lesion that threatens the structural and functional integrity of the genome. In this thesis the repair of DSBs was investigated using the bacterium Escherichia coli, which repairs DNA breaks almost exclusively by homologous recombination. The studies described focus on the repair of damage induced by reactive oxygen species, but especially on the RecN protein, which is associated specifically with the repair of double-strand breaks.

The RecN protein is highly conserved across bacterial species and in E. coli has been identified as a key factor in the repair of DNA breaks. In this thesis three RecN homologs were analysed. RecN from Haemophilus influenzae is shown to be capable of replacing the functions of E. coli RecN in vivo. However, homologs from Aquifex aeolicus and Bacteriodes fragilis cannot do so.

Biochemical analysis of all three RecN homologs was undertaken. The H. influenzae RecN and A. aeolicus RecN were shown to have weak ATPase activity and an ability to interact with single-stranded DNA. ATPase deficient mutants of the RecN proteins were created and used to demonstrate the functional importance of the ATP hydrolysis. In the case of E. coli and H. influenzae, the ATPase defective mutants failed to function in vivo. In vitro, the ATPase deficient H. influenzae RecN mutant and a similar mutant of A. aeolicus RecN failed to interact with single-stranded DNA. These data are discussed in terms of a relationship between RecN and the structural maintenance of chromosome family of proteins.

Finally, a model for RecN activity is presented based on those developed to explain the function of structural maintenance of chromosome proteins and the new data presented here. In this model, RecN is suggested to trap DNA molecules holding a break site and repair template in close proximity, facilitating the repair of DNA breaks by homologous recombination. The possibility of RecN acting as a global, damage induced cohesin is also discussed.

Item Type:Thesis (PhD)
Supervisors:Lloyd, R.G.
Uncontrolled Keywords:Recombiantion, DNA repair, SMC, OxyR, AhpC, Oxidative stress.
Faculties/Schools:UK Campuses > Faculty of Medicine and Health Sciences > School of Biology
ID Code:685
Deposited By:Mr Stuart R Wood
Deposited On:04 Nov 2009 13:31
Last Modified:04 Nov 2009 13:31

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