<i>Escherichia coli </i>RecA is a versatile protein that is involved in homologous recombination, and coordination of both the DNA damage response and translesion synthesis. Single-stranded DNA (ssDNA) that is generated at the site of double-stranded breaks serves as a signal to activate RecA. This allows RecA to form a long helical filament on the ssDNA, which is required in recombination, hydrolysis of ATP, and mediating the self-cleavage of some ser-lys dyad proteins such as the LexA repressor. In this thesis, the formation of the RecA/LexA complex did not require preactivation by ssDNA, instead a volume excluding agent in the presence of LexA was able to stimulate its formation. These preliminary results led to a hypothesis that the formation of the RecA/LexA complex is a thermodynamic process that involves three steps: (1) a change in RecAs conformation towards the active form, (2) a change in LexAs conformation towards the cleavable form (i.e. burial of the ser-lys dyad catalytic residues), and (3) the binding between the active form of RecA and the cleavable form of LexA. Evidence for this model was shown by the ability of either NaCl, LexA K156A, an ATP substrate, or a volume excluding agent to enhance the stability of the RecA/LexA complex, which was detected by both the ATPase and coprotease assays. Hyper-active RecA mutants, isolated form the yeast two-hybrid screen, were also tested, however they did not enhance the stability of the complex. Additionally, RecAs binding preference for the monomer or dimer form of LexA was examined, since it is unknown which species of LexA is able to enhance the stability of the complex. To generate the monomer form of LexA, single point mutations were introduced at the dimer interface of the protein such that its dimerization was disrupted by charge-charge repulsions. Based on the inhibition assay, RecA was found to bind preferentially to dimer form and not the monomer form of LexA, possible reasons for these results are discussed.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-08182006-191437 |
| Date | 28 August 2006 |
| Creators | Moya, Ignace Adolfo |
| Contributors | Roesler, William J., Luo, Yu, Khandelwal, Ramji L., Geyer, C. Ronald, Bull, Harold |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-08182006-191437/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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