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.

Affinity isolation and mass spectral analysis of 1,10-phenanthroline (OP)-stimulated UV-damaged-DNA binding proteins expressed in zebrafish (Danio rerio) embryos

“…In this study, UV irradiation was carried out by pipetting the duplex TC probe (1 pmol/1 ll in 10 mM Tris-HCl and 1 mM EDTA) onto a piece of parafilm and irradiated with UV in an XL-1500 UV crosslinker (Spectronics, Westbury, NY, USA). The specific production of a 6-4PP on UV-irradiated TC probe was confirmed in our previous studies (Lai et al 2006). …”

“…5b). Due to the absence of the 30-and 35-kDa UV-damaged-DNA binding factors recognized by the same anti-zfVg1 antibody (Lai et al 2006) in fraction 12, novel DNA damage recognition proteins unreactive to the anti-zfVg1 antibody targeting the Lv1 domain were predicted to generate the 6-4PP-binding activity stimulated by OP.…”

“…After blocking of nonspecific binding sites in Tris buffer (pH 7.5) containing 5% (w/v) skimmed milk and 1% (w/v) BSA, the membrane was incubated at room temperature for 1 h with a mouse monoclonal antibody (JE-8D6, Biosense Laboratories, Bergen, Norway) (1:1,000 dilution in the blocking buffer) targeting the Lv1 domain of zfVg1. The same antibody was shown to recognize the 30-and 35-kDa 6-4PP-binding polypeptides isolated in our previous study (Lai et al 2006). The presence of zfVg1-Lv1-like proteins was detected by the addition of a horseradish peroxidase-conjugated second antibody and chemiluminescent enzyme substrates.…”

“…The extracts of 12 hpf zebrafish embryos were prepared using CHAPS lysis buffer as described in our earlier reports (Hsu et al 2002;Lai et al 2006). Zebrafish extract proteins (8 mg) were separated by isoelectrofocusing (IEF) between pH 3 and 10 for 6 h at 4°C using a preparative IEF system (Rotofor cell, Bio-Rad Laboratories, Richmond, CA).…”

“…The two polypeptides were shown to contain amino acid sequences present predominantly in the N-terminal lipovitellin 1 (Lv1) domain of the 150-kDa zebrafish vitellogenin 1 (zfVg1) (GenBank accession No. NP_001038362) when analyzed by peptide mass fingerprinting (PMF) and MS/MS (Lai et al 2006). In this study, UV-damaged-DNA binding proteins in zebrafish extracts were fractionated by isoelectrofocusing and both OP-sensitive and OP-stimulated fractions were detected.…”