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Title: Affinity isolation and mass spectral analysis of 1,10-phenanthroline (OP)-stimulated UV-damaged-DNA binding proteins expressed in zebrafish (Danio rerio) embryos
Authors: Yi-Show Lai;Feng-Ju Hsieh;Todd Hsu
Contributors: 國立台灣海洋大學:生命科學系
Keywords: Embryos;DNA damage;Protein;UV;Vitellogenin;Zebrafish
Date: 2012-08
Issue Date: 2016-04-11T05:38:06Z
Publisher: Fish Physiology and Biochemistry
Abstract: 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.
Relation: 38(4),pp.1117-1129
URI: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/37673
Appears in Collections:[生命科學系] 期刊論文

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