碩士 / 國立臺灣海洋大學 / 生物科技研究所 / 93 / Distorted DNA helical structures induced by covalent chemical adducts or UV-irradiation are removed by specific DNA repair pathways and these repair systems are initiated by the binding of damage-recognition protein to DNA lesions. Distorted DNA-binding proteins in lower plants are studied in this thesis by electrophoresis mobility shift assay (EMSA), affinity adsorption, 2-D gle electrophoresis and mass spectrometric analysis using the unicellular alga Chlorella pyrenoidosa as a model system. Some proteins in C. pyrenoidosa extracts bound preferentially to oligonucleotides carrying 6-4PPs based on EMSA. After fractionating the algal extracts through a pH gradient from 3 to 10 by preparation isoelectrofocusing, (6-4)PPs-binding proteins having pIs between 3.0 and 7.3 were detected. Damage-recognition proteins recognizing both (6-4)PPs and cisplatin-induced intrastrand GG crosslinking were found in fractions with pH 3, 5.5, 7.2. The preferential binding of these proteins to different types of DNA damage suggested their possibility as proteins involved in the damage recognition step of the wide-spectrum pathway of nucleotide excision repair.
Cell-free extracts of C. pyrenoidosa were preferentially captured by immobilized cisplatin-damaged DNA in affinity adsorption. Three polypeptides with molecular weights between 60.4 to 86.4 kDa and pIs between 5.5 to 7.1 were detected after 2-D electrophoresis using IPG strips containing pH gradients from 5 to 8, and each with a 2 to 3 stronger affinity for distorted helical structures. More over, the pIs range of these three polypeptides were very similar to the preparation isoelectrofocusing fractions which contain (6-4)PPs and cisplatin-induced intrastrand GG crosslinking binding proteins. Suggested those three polypeptides were involved in the damage recognition step of the wide-spectrum pathway of nucleotide excision repair. After mass spectrometric analysis of cisplatin-damaged DNA binding polypeptides. The 63.8 kDa polypeptide that showed to no cisplatin-damaged-dependent binding was homolog of the Scenedesmus quadricauda rubisco large subunit. And the other cisplatin-damaged-DNA binding proteins mass spectrometric analysis are underway.
| Identifer | oai:union.ndltd.org:TW/093NTOU5111017 |
| Date | January 2005 |
| Creators | Pei-San Lee, 李佩珊 |
| Contributors | Todd Hsu, 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 | 69 |
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