Genome stability has great importance in maintaining cell viability and optimal functionality of cellular processes. Loss of genome stability can lead to cell death in the simplest organisms and to deregulation of the cell proliferation machinery in higher organisms, potentially causing cancer or morbid states. The Single Stranded DNA Binding (SSB) protein of Escherichia coli is an essential protein that binds and stabilises ssDNA stretches. Its role is particularly crucial during DNA replication, recombination and repair processes and it has therefore been predicted to play a prominent role in the maintenance of genome stability. The role of SSB in genome instability was investigated using an E. coli strain in which, the expression of the ssb gene was placed under the control of the arabinose promoter. The level of SSB protein present in the cell could therefore be tuned by varying the arabinose concentration in the medium. A wide characterisation of the behaviour of the strain at low SSB level was carried out. Viability and growth tests showed that a threshold level of protein is required to allow normal growth. Microscopy analyses were carried out to follow cell division, nucleoid morphology and SOS response activation. Cells grown at low SSB level, showed a phenotype consistent with impaired cell division and altered nucleoid morphology. The SOS response was activated at low SSB levels and cell elongation was detected. Lowering the arabinose concentration in solid medium allowed the selection of suppressor strains that could form colonies under the new conditions. Sequencing of the entire genome of one such suppressor strain was carried out revealing a possible candidate for the phenotype change. The stability of a 105bp and of a 246bp DNA imperfect palindromes and the stability of CAG·CTG trinucleotide repeat arrays, inserted in the E. coli chromosome, were investigated in correlation to the SSB cellular level. Lowering the SSB level in cells grown on solid medium, increased the instability of the 105bp palindrome presumably by increasing the number of slippage events. On the other hand, SSB overexpression did not have an effect on the stability of the 246bp palindrome. The stability of a leading strand (CAG)75 repeat array was highly increased by overexpressing SSB, while the same effect was not observed for a leading strand (CTG)137 repeat array. Furthermore, excess SSB caused a change in the deletion size distribution profile for the leading strand (CAG)75 strain, lowering the bias towards big deletions. This is consistent with SSB being able to preferentially impede the formation of big DNA hairpins. Also, SbcCD nuclease was shown to have an effect on the deletion size distribution profile of the leading strand (CTG)137 strain. The lack of SbcCD led to a slight reduction of the number of big deletions.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:562547 |
| Date | January 2009 |
| Creators | Andreoni, Federica |
| Contributors | Leach, David. ; Poon, Wilson |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/3993 |
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