Affinity isolation and mass spectral analysis of 1,10-phenanthroline (OP)-stimulated UV-damaged-DNA binding proteins expressed in zebrafish (Danio rerio) embryos
- Authors
- Lai, Y.S., Hsieh, F.J., and Hsu, T.
- ID
- ZDB-PUB-120125-13
- Date
- 2012
- Source
- Fish physiology and biochemistry 38(4): 1117-1129 (Journal)
- Registered Authors
- Hsu, Todd
- Keywords
- embryos, DNA damage, protein, UV, vitellogenin, zebrafish
- MeSH Terms
-
- Animals
- Chelating Agents/pharmacology
- DNA/metabolism
- DNA Damage
- DNA-Binding Proteins/antagonists & inhibitors
- DNA-Binding Proteins/metabolism*
- Electrophoresis, Gel, Two-Dimensional
- Embryo, Nonmammalian/metabolism*
- Embryo, Nonmammalian/radiation effects
- Female
- Immunoblotting
- Isoelectric Focusing
- Male
- Mass Spectrometry
- Phenanthrolines/pharmacology
- Pyrimidine Dimers/metabolism
- Ultraviolet Rays*
- Zebrafish/embryology
- Zebrafish/metabolism*
- PubMed
- 22252336 Full text @ Fish Physiol. Biochem.
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.