Shen Yung-Chi, Hsu Todd, Ling Li-Bin, You Wen-Chian, Liu Chia-Wei
Department of Internal Medicine, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Keelung, 204, Taiwan.
Department of Bioscience and Biotechnology and Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan.
Fish Physiol Biochem. 2017 Apr;43(2):663-676. doi: 10.1007/s10695-016-0321-4. Epub 2017 Jan 10.
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 T 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.
核苷酸切除修复（NER）可去除扭曲螺旋的DNA损伤，如紫外线诱导的嘧啶二聚体和顺铂诱导的链交联。我们早期的研究已鉴定出与150 kDa卵黄生成素1（Vg1）同源的低分子量蛋白质，作为在发育中的斑马鱼（Danio rerio）中表达的紫外线损伤DNA结合因子。本研究探讨了Vg1样蛋白是否也参与斑马鱼的NER。对受精后12小时（hpf）斑马鱼提取物蛋白进行亲和捕获的免疫印迹分析显示，一种30 kDa的Vg1样多肽与紫外线损伤的DNA存在瞬时结合。基于转录的体外修复试验表明，在用12 hpf胚胎或96 hpf幼虫的提取物孵育受损质粒后，由SP6 RNA聚合酶调节启动子驱动的标记cDNA的UVC或顺铂抑制转录活性显著上调。在存在单克隆抗斑马鱼Vg1抗体的情况下，紫外线或顺铂抑制的转录上调被消除。12和96 hpf斑马鱼提取物中紫外线诱导修复对外源ATP的不同敏感性表明Vg1样NER因子的表达具有发育依赖性。T核酸内切酶V消化试验表明，抗Vg1抗体对紫外线诱导的环丁烷嘧啶二聚体的切除没有抑制作用。我们的结果确定了Vg1样因子参与发育中的斑马鱼的NER，并且这些因子可能在NER的切口后步骤发挥作用。
