碩士 / 國立臺灣海洋大學 / 生命科學暨生物科技學系 / 105 / Nucleotide excision repair (NER) is the main pathway to remove the UV-induced bulky DNA lesions like cyclobutane pyrimidine dimer (CPD) and 6-4 photoproduct (6-4PP). Our earlier studies showed the ability of ROS-inducing agents cadmium (Cd) and paraquat (PQ) to down-regulate CPD binding activities in zebrafish embryos as shown by band shift assay. Exposure of embryos to Cd and PQ, however, caused a weak stimulation of 6-4PP binding activity. The objective of this study was to further explore the differential effects of oxidative stressors on CPD and 6-4PP sensing activities and the possible mechanisms. Exposure of zebrafish embryos at 1 h post fertilization (hpf) to PQ at 30 to 200 μM for 9 hr did not affect 6-4PP binding activity, while hydrogen peroxide (HP) at 200 μM and Cd at 5 μM caused a weak inhibition of this binding activity. The formation of CPD band shifts was inhibited by HP, Cd and PQ at all levels. Hence, HP, Cd and PQ imposed differential inhibitory effects on UV-damaged-DNA binding activities.
UV-damaged DNA-binding protein (UV-DDB) and xeroderma pigmentosum C (XPC) are damage recognition factors of NER. Down-regulated gene expression of DDB2 determined by real-time RT-PCR was partially consistent with inhibition of CPD binding activity when zebrafish embryos were exposed to HP at 100 μM and PQ at 30 to 100 μM. No close association between inhibition of 6-4PP binding activity and XPC gene expression was observed in embryos exposed to 5 μM Cd.
Participation of low-molecular-weight homologs of the 150-kDa zebrafish vitellogenin1 (Vg1) in NER has been identified in our previous studies. To explore the possible involvement of Vg1-ike protein in the damage recognition stage of NER, UV crosslinking was conducted to examine if Vg1-like factors contacted UV-damaged DNA. Crosslinking assay showed no changes in the molecular weights of Vg1-like proteins based on immunoblot analysis of Vg1-like proteins present in zebrafish extracts containing UV-irradiated or unirradiated DNA probe. No crosslinking of low-molecular-weight Vg1-like factors to CPDs or 6-4PPs was observed under oxidative stress. How Vg1-like factors participate in NER needs further study.
| Identifer | oai:union.ndltd.org:TW/105NTOU5613018 |
| Date | January 2017 |
| Creators | Zhuang, Jie, 莊婕 |
| Contributors | Hsu, Todd, 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 | 38 |
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