| Summary: | 碩士 === 國立海洋大學 === 水產生物技術研究所 === 88 === A strong UV-damaged-DNA binding activity was detected in the extracts of zebrafish early embryos ( 3 to 12 hrs after fertilization) The extracts prepared from zebrafish at developmental stages from 12 to 84 hr showed a gradual decrease in binding affinity toward UV-damaged DNA. The extracts of 3 to 12-hr-old zebrafish displayed a preferential binding to a duplex oligonucleotide containing either a cyclobutane pyrimidine dimer (CPD) or a (6-4)photoproduct (6-4 PP), and both types of distorted DNA structures were recognized by the same binding activity. The molecular mass of the primary extract protein-CPD complex formed after UV crosslinking was found to decrease along with zebrafish development, indicating a development-dependent expression of damage-recognition proteins. The binding activity found in 12-hr-old embryos contained at least two protein fractions that could be eluted from a DEAE-cellulose column by NaCl at 0.1 M and at 0.2 M. Both eluates showed a similar level of preferential binding to CPD, while the 0.1 M NaCl eluate bound much more strongly to 6-4 PP than the 0.2 M NaCl eluate, particularly at a lower protein level. Crosslinked complexes with molecular weights about 100,38, and 34 Kda were found by SDS-PAGE after the interaction between the 0.1 M NaCl eluate and a 6-4 PP, but the 34 Kda complex was not found for the 0.2 M NaCl eluate. Since 6-4 PP is a strong DNA-distorting agent, the polypeptide contained in the 34 Kda complex was therefore regarded as a key factor in recognizing distorted DNA. The function of UV-damaged-DNA binding proteins detected in zebrafish early embryos remains to be answered, yet they may participate in the process of excision repair as damage-stimulated repair synthesis of UV-irradiated plasmid DNA was more apparent in 12-hr-old zebrafish extracts than in those of older zebrafish.
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