title: Affinity isolation and mass spectral analysis of 1,10-phenanthroline (OP)-stimulated UV-damaged-DNA binding proteins expressed in zebrafish (Danio rerio) embryos.
abstract: Our earlier studies indicated the high expression of a UV-damaged-DNA binding activity in zebrafish (Danio rerio) embryos at 12 h postfertilization (hpf). Two 30- to 35-kDa polypeptides homologous to the N-terminal lipovitellin 1 (Lv1) domain of the 150-kDa zebrafish vitellogenin 1 (zfVg1) were identified as the damage recognition factors in zebrafish extracts, and the metal-chelating agent 1,10-phenanthroline (OP) was found to inhibit the embryonic UV-damaged-DNA binding activity. This study further explored the DNA damage-sensing components in 12 hpf zebrafish extracts. UV-damaged-DNA binding proteins were enriched from zebrafish extracts by isoelectrofocusing. Both OP-sensitive and OP-stimulated, UV-damaged-DNA binding activities were detected in fractionated zebrafish extracts. Two-dimensional gel electrophoresis of proteins captured by an immobilized oligonucleotide carrying a UV-induced (6-4)photoproduct (6-4PP) revealed a 25-kDa polypeptide as the major 6-4PP-binding factor in an OP-stimulated fraction. Three 25-kDa factors that bound weakly to 6-4PPs were also isolated. The four polypeptides having pIs between 7.0 and 7.3 were unreactive to an anti-zfVg1 antibody targeting the Lv1 domain. Mass spectral analysis showed the appearance of amino acid sequences LPIIVTTYAK and IPEITMSK in all 25-kDa polypeptides and sequences exactly matching those contained in the four factors exist only in the C-terminal Lv2 domain of zfVg1, reflecting the origination of these factors from enzymatic cleavage of the Lv2 domain at slightly different positions. The OP-stimulated fraction produced a much stronger UV-dependent DNA incision activity in the presence than in the absence of OP, suggesting the association of these factors with DNA damage repair under metal-deficient conditions.
authors.affiliation: Institute of Bioscience and Biotechnology, Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, No. 2, Pei-Ning Rd., Keelung, 20224, Taiwan, ROC.
authors.forename: Yi-Show
authors.initials: YS
authors.lastname: Lai
authors.affiliation: Institute of Bioscience and Biotechnology, Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, No. 2, Pei-Ning Rd., Keelung, 20224, Taiwan, ROC.
authors.forename: Feng-Ju
authors.initials: FJ
authors.lastname: Hsieh
authors.affiliation: Institute of Bioscience and Biotechnology, Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, No. 2, Pei-Ning Rd., Keelung, 20224, Taiwan, ROC. toddhsu@mail.ntou.edu.tw.
authors.forename: Todd
authors.initials: T
authors.lastname: Hsu
mesh_terms: D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004625:Embryo, Nonmammalian / Q000378:metabolism* / Q000528:radiation effects; D005260:Female; D015151:Immunoblotting; D007525:Isoelectric Focusing; D008297:Male; D013058:Mass Spectrometry; D010618:Phenanthrolines / Q000494:pharmacology; D011740:Pyrimidine Dimers / Q000378:metabolism; D014466:Ultraviolet Rays; D015027:Zebrafish / Q000196:embryology / Q000378:metabolism*
chemical_list: D002614:Chelating Agents; D004268:DNA-Binding Proteins; D010618:Phenanthrolines; D011740:Pyrimidine Dimers; C058406:pyrimidine-pyrimidone dimer; D004247:DNA; C025205:1,10-phenanthroline