Within all cells, complex molecular systems exist that are responsible for maintaining genome stability by detecting and repairing dangerous alterations in DNA. Ensuring the accurate and efficient functioning of such systems is necessary for the preservation of DNA integrity and avoidance of disease. The flexible and diverse modes of DNA-binding exhibited by human p53 permits this ‘guardian of the genome’ to elicit versatile cellular activities that are crucial in monitoring threats to genome dynamics and conducting appropriate responses. In conjunction with its sequence-specific DNA-binding activity that is essential to target gene transactivation, p53 can bind to unusual DNA structures independent of DNA sequence and it has been proposed this activity may allow p53 to interact with detrimental secondary structures that arise in unstable genomic regions. To provide further insight into p53-DNA interactions, an in vitro DNA binding assay was developed that was used to characterise binding properties towards several DNA molecules to allow comparison of non-specific, sequence-specific and structurespecific binding. It was determined that unusual structures in DNA significantly enhanced p53 binding in non-sequence specific DNA and that the presence of internal hairpin regions induced binding comparable to sequence-specific binding. In vivo p53-DNA interactions were also quantified using chromatin immunoprecipitation and variations in preference to different response element sequences was ascertained. DNA binding is also central to the ability of Ku proteins to function as essential components of non-homologous end joining and telomere maintenance in eukaryotes. Prokaryotic homologues of Ku proteins that function as homodimers in two-component repair systems have also been identified. Recently, 3 Ku homologues in Streptomyces coelicolor were reported, but very little is currently known regarding their biological activity. It was discovered that all 3 Ku proteins exhibited varied independent DNA-binding properties that were influenced by DNA topology, size and end-structure. Unusually for Ku, it was found 1 of these proteins exhibited strong binding to single-stranded DNA. Precipitation assays determined that these proteins may act as DNA end synapsis mediators during the DNA endjoining process and ligation experiments revealed Ku was responsible for rigidifying DNAs or completely inhibiting ligation activity, probably via DNA end-protection activity. Experimental evidence indicated that specific interactions could occur between S. coelicolor Ku suggesting these proteins form both homodimers and heterodimers.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:569316 |
| Date | January 2010 |
| Creators | Cobb, Andrew Martin |
| Publisher | University of East Anglia |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://ueaeprints.uea.ac.uk/10586/ |
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