Fish Physiology and Biochemistry

, Volume 43, Issue 2, pp 663–676 | Cite as

Identification of low-molecular-weight vitellogenin 1 (Vg1)-like proteins as nucleotide excision repair (NER) factors in developing zebrafish (Danio rerio) using a transcription-based DNA repair assay

  • Yung-Chi Shen
  • Todd Hsu
  • Li-Bin Ling
  • Wen-Chian You
  • Chia-Wei Liu


Nucleotide excision repair (NER) removes helix-distorting DNA lesions such as UV-induced pyrimidine dimers and cisplatin-induced strand crosslinking. Our earlier studies have identified low-molecular-weight proteins homologous to the 150-kDa vitellogenin 1 (Vg1) as UV-damaged DNA-binding factors expressed in developing zebrafish (Danio rerio). This present study explored if Vg1-like proteins also participated in NER in zebrafish. Immunoblot analysis of affinity-captured 12 h post-fertilization (hpf) zebrafish extract proteins showed a transient binding of a 30-kDa Vg1-like polypeptide to UV-damaged DNA. A transcription-based in vitro repair assay revealed a significant up-regulation of UVC or cisplatin-suppressed transcriptional activity of a marker cDNA driven by a SP6 RNA polymerase-regulated promotor after incubating the damaged plasmid with the extracts of 12 hpf embryos or 96 hpf larvae. The up-regulation of UV or cisplatin-suppressed transcription was abolished in the presence of a monoclonal anti-zebrafish Vg1 antibody. The differential sensitivity of UV-induced repair in 12 and 96 hpf zebrafish extracts to exogenous ATP suggested a development-dependent expression of Vg1-like NER factors. A T4 endonuclease V digestion assay showed no inhibition of the anti-Vg1 antibody on the excision of UV-induced cyclobutane pyrimidine dimers. Our results identified the participation of Vg1-like factors in NER in developing zebrafish, and these factors may function at post-incison steps of NER.


Cisplatin Nucleotide excision repair Protein UV Vitellogenin Zebrafish 



This work was financially supported by the Ministry of Science and Technology of Taiwan, Republic of China, under grant numbers NSC 100-2313-B-019-009 and NSC 102-2313-B-019-017.

Supplementary material

10695_2016_321_MOESM1_ESM.pptx (125 kb)
Suppl. Fig.1. Detection of T4 endonuclease V-mediated incision of UV-irradiated TT probe carrying a CPD by denaturing PAGE. Biotin-labelled duplex TT probe (4 pmole) was irradiated with UV at 9 kJ m-2 to generate a CPD between two adjacent TT bases on one strand according to a previous report (Pasheva et al. 1998). To confirm the production of CPD, UV-irradiated or unirradiated TT probe was incubated at 37 °C for 16 h with CPD-specific T4 endonuclease V(1 unit). The reaction mixture was electrophoresed on a 6% denaturing polyacrylamide gel and blotted overnight onto a nylon membrane. The presence of a biotin-labelled 5’ short single strand fragment (see arrow) due to enzyme-mediated incision and strand cleavage was detected by incubating the membrane with streptavidin-conjugated alkaline phosphatase and chemilluminesent enzyme substrates. (PPTX 124 kb)


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Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • Yung-Chi Shen
    • 1
    • 2
  • Todd Hsu
    • 2
  • Li-Bin Ling
    • 2
  • Wen-Chian You
    • 2
  • Chia-Wei Liu
    • 2
  1. 1.Department of Internal Medicine, Chang Gung Memorial HospitalChang Gung University College of MedicineKeelungTaiwan
  2. 2.Department of Bioscience and Biotechnology and Center of Excellence for the OceansNational Taiwan Ocean UniversityKeelungTaiwan

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