Low-molecular-weight vitellogenin 1-like proteins are components of a UV-damaged-DNA binding activity highly expressed in zebrafish (Danio rerio) embryos

Abstract

A strong UV-damaged-DNA binding activity had been detected in the extracts of zebrafish embryos at 12 hr after fertilization by gel shift assay (Hsu et al. 2002. Fish Physiol Biochem 25:41-51). We attempted to study the components of this binding activity and their importance in DNA damage recognition. Among the proteins extracted from gel retardation complexes, a 30- and a 35-kDa polypeptide binding preferentially to 6-4photoproducts (6-4PPs) generated by UV irradiation were identified by peptide mass fingerprinting (PMF) as homologs of zebrafish vitellogenin I (zfVg1), a 150-kDa metalloprotein known as the precursor of yolk proteins in embryos. zfVg1-like polypeptides ranging from 25 to 105 kDa were detected in 12- and 96-hr-old zebrafish extracts by immunoblot analysis. Immunoblot analysis of affinity-captured proteins confirmed the preferential binding of the 30-35-kDa polypeptides to the 6-4PP probe, while 96-hr-old larval extracts containing very low levels of these two factors failed to recognize 6-4PPs. The presence of zfVg1-like factors was important in maintaining the embryonic UV-binding activity, as inclusion of a monoclonal anti-zfVg1 antibody in reaction mixtures caused a concentration-dependent reduction in 6-4PP-specific binding. In contrast, DNA damage recognition was not disturbed at all by an anti-HSP 70 antibody. The formation of 6-4PP-binding complexes was abolished after the addition of the metal chelating agent 1,10-phenanthroline (OP) to zebrafish extracts and the loss of UV-binding capacity correlated with the disappearance of the 35-kDa factor in OP-treated extracts. Our results demonstrated the ability of low-molecular-weight zfVg1-like proteins in zebrafish embryos to bind UV-damaged DNA and the expression of this embryonic UV-binding activity was metal dependent. Whether zfVg1-like UV-binding proteins are involved in repairing damaged DNA in embryos or in processing helical structures similar to UV-distorted DNA needs further investigation.