In an effort to understand the molecular mechanisms of DNA alkylation repair, I initiated molecular cloning of a novel gene, ' NGS1'. The <i>Saccharomyces cerevisiae ngs1-1</i> mutant was previously identified by its enhanced sensitivity to simple DNA alkylating agents such as methyl methanesulfonate but not to UV. Molecular cloning and sequencing of 'NGS1' as a putative DNA alkylation repair gene revealed that it is identical to 'MRE11'/'RAD58', a gene that is involved in meiotic recombination and DNA recombinational repair. In order to investigate functional domains of the Mre11 protein, I determined the nucleotide sequence alterations of a number of 'mre11' mutant alleles, including 'ngs1-1', 'mre11-1' ('ts'), 'mre11-2', 'mre11-3' and 'mre11-58'. The location of various 'ngs1'/' mre11'/'rad58' mutations combined with the deletion analysis indicates that the functional domain(s) resides in the highly conserved N-terminus of Mre11. I also investigated various roles of Mre11 in spontaneous and DNA damage-inducedmitotic recombination. The assays used in this study show that the <math> <f> mre<it>11<g>D</g></it></f> </math> mutation enhances inter-chromosomal recombination but decreases the intra-chromosomal deletion frequency. In addition, 'MRE11' appears to play different roles during spontaneous and alkylation-induced homologous mitotic recombination. Physical interactions between members of the 'RAD52' epistasis group have been detected genetically and biochemically. These protein interactions also appear to be important at the early stage of meiotic recombination. Mre11 has been shown to interact with itself, Rad50 and Xrs2 in a yeast two-hybrid system. Preliminary studies employing deletion analysis predicted that the self-interaction domain of Mre11 resided at the N-terminus of the protein. To determine domain(s) required for Mre11 complex formation, and to elucidate the relationship between this complex formation and 'MRE11' DNA repair function, I employed a combined yeast two-hybrid and functional analyses. My results indicate that both Mre11 dimerization and interaction with Rad50 are essential for recombinational repair. I found that the N-terminus of the Mre11 protein constitutes the core homodimerization and heterodimerization domain and is sufficient for Mre11 DNA repair activity. Collectively, these studies support the hypothesis that Mre11 self-association as well as its assembly into a multi-protein complex consisting of Mre11 and Rad50 are essential for effective DNA recombinational repair. Using the sequence of yeast 'MRE11', isolated the full-length ' hMRE11B' cDNA from a human HeLa cell cDNA library. Compared to the previously identified 'hMRE11', 'hMRE11B' contains an additional 84 base pair sequence that results in a 28 amino acid insertion close to the C-terminus. Overexpression of 'hMRE11B' does not complement the alkylation sensitivity of the 'mre11' null and temperature sensitive mutant strains. My results suggest that species-specific protein interaction determines the functional specificity of 'MRE11 ' and that the participation of the C-terminus of Mre11 protein plays an important role in this regard. (Abstract shortened by UMI.)
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-10212004-001328 |
| Date | 01 January 1999 |
| Creators | Chamankhah, Mahmood |
| Contributors | Xiao, Wei |
| 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-10212004-001328 |
| 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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