Recognition and incision of UV-damaged DNA by vitellogenin1-like proteins in zebrafish (Danio rerio) early embryos

• Main Authors: Feng-Ju Hsieh, 謝豐如
• Other Authors: Todd Hsu
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/99556250569472509731

碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 94 === ABSTRACT Dipyrimidine photoproducts induced by UV irradiation on DNA are primarily removed from UV-damaged DNA by nucleotide excision repair (NER). NER proteins homologous to the human XPA were not detected in the extracts of zebrafish (Danio rerio) embryos, yet these extracts were previously found to contain two 30 to 35 kDa UV-damaged-DNA binding proteins homologous to the 150 kDa vitellogenin1 (Vg1). The objectives of this research were to examine if multiple Vg1-like UV-binding factors existed in zebrafish embryos and whether these factors were involved in NER. Nonspecific and UV-specific DNA binding molecules present in 12-hr-old zebrafish embryos were efficiently separated by a reparative isoelectrofocusing. Two forms of Vg1-like UV-binding proteins either sensitive or resistant to the metal chelating agent 1, 10-phenanthroline (OP) were identified in the extracts of zebrafish early embryos. The binding of weak acidic proteins in OP-sensitive fractions to (6-4) -photoproducts (6-4PPs) was significantly inhibited by metal chelation, and this inhibition correlated well with the instability of the 35 kDa Vg1-like polypeptide. In contrast, 6-4PP-specific binding produced by proteins having pIs about 7 to 8 in OPresistant fractions was resistant to and often stimulated by metal chelation. Four 25 kDa Vg1-like DNA damage-recognition polypeptides had been isolated from an OP-stimulated fraction. Using a supercoiled plasmid as a DNA repair substrate, the extracts of zebrafish embryos were able to induce a damage-specific DNA incision in the presence of ATP, generating a UV-dependent increase in the level of openform plasmid. As the level of UV-independent DNA incision was increased by the presence of an anti-Vg1 antibody, Vg1-like UV-binding proteins might be the damage-recognition factors in embryonic NER. A repair system composed of multiple proteins in zebrafish extracts was believed to induce the UVdependent DNA incision, since each protein fraction obtained after isoelectrofocusing showed a much weaker cutting effect than the crude extracts.