碩士 / 國立海洋大學 / 水產生物技術研究所 / 90 / Abtract
Mismatch repair (MMR) is a DNA repair pathway that removes DNA mispairs or insertion-deletion loops generated during replication, recombination or spontaneous deamination of 5-methylcytosine. The mechanisms MMR in plants are still unclear and the unicellular alga (Chlorella pyrenoidosa) was used in this research as a model system to study MMR proteins in plants. The extracts of C. pyrenoidosa showed a strong preferential binding to a hetroduplex probe carrying a G-T or a C-T mispair as determined by electrophoretic mobility shift assays (EMSA). Algal extract proteins bound weakly to C-C mispair and intermediate binding efficiencies were observed for G-A and G-G mispairs. Addition of KCl up to 1M to EMSA mixtures did not affect the binding of C.pyrenoidosa extracts to mismatched DNA. High-shifting binding complexes with loose structures were formed after incubating the algal extracts and the heteroduplex probes in the presence of 0.75 and 1.5mM ATP. A UV-damaged duplex probe carrying either a cyclobutane pyrimidine dimer or a 6-4 photoproduct was unable to compete the binding of the G-T probe to C. pyrenoidosa extracts, indicating the specificity of mismatch recognition proteins in C.pyrenoidosa to their binding targets.
SDS-PAGE analysis and silver staining of the proteins eluted from G-T and homoduplex DNA binding complexes revealed the presence of several mismatch-binding polypeptides with molecular weights between 15 and 40KDa. One 18KDa G-T binding polypeptide with very low affinity for the homoduplex probe was excised from the gel for MALDI-TOF (Matrix Assisted Laser Directed Ionization-Time of Flight), and the mass spectrum of this polypeptide showed no homology with that of any protein stored in the Mascot data bank, suggesting that the 18KDa polypeptide was a novel mismatch binding protein.
The algal extracts were found to contain a weak repair activity as shown by in vitro repair assay after incubation with bacteriophages inserted with a heteroduplex fragment containing a nick at the 5`end of DNA mismatches. The repair efficiency was very limited because of nonspecific nucleolytic activities present in the algal extracts. Inclusion of polydI;dC in repair reaction mixtures slightly improved the repair efficiency and repair substrates suitable for algal extract proteins must be developed.
| Identifer | oai:union.ndltd.org:TW/090NTOU0613006 |
| Date | January 2002 |
| Creators | 阮婷儀 |
| Contributors | 許濤 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 81 |
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