Isolation and characterization of a protein participating in nucleotide excision repair in human cells.

紫外线损伤特异性结合蛋白被认为在紫外线照射的人细胞DNA修复的早期步骤中起重要作用。我们观察到核蛋白和细胞质蛋白选择性地与紫外线照射的DNA结合。我们纯化了一种从核提取液到人体的结合因子，命名为NF-10。其相对分子质量为-40 kDa。使用针对环丁烷嘧啶二聚体和(6-4)光解产物的光解酶实验表明，NF-10蛋白通过(6-4)光解产物与UV-DNA结合，利用针对NF-10蛋白的单抗，我们可以证明所有的着色性干皮病互补组细胞系中都存在NF-10蛋白。这一结果表明，NF-10是一种不同于已报道的XPA蛋白和UV-DNA结合因子的新蛋白。该抗体去除细胞提取液中的核因子-10蛋白，使其体外修复活性降低，但加入该蛋白可恢复其活性。该蛋白的部分肽序列与人3-磷酸甘油醛脱氢酶(GAPD)的部分序列几乎相同。纯化的核因子-10蛋白在糖酵解中发挥GAPD活性。这些结果表明，Gapd可能在核苷酸切除修复和糖酵解中发挥作用，可能是在损伤识别过程中。

UV-damage-specific binding proteins are considered to play important roles in the early steps of DNA repair in human cells irradiated with UV.We have survayd the nuclear and cytoplasmic proteins binding selectively to UV-irradiated DNA.We purifiied one of the binding factors from nuclear extracts to humogeneity, which was designated NF-10. Its molecular weight is - 40kDa. The experimets using photolyases specific for cyclobutane pyrimidine dimer and (6-4) photoproduct revealed that the NF-10 protein binds to UV-DNA though (6-4) photoproducts.By using a monoclonal antibody raised against the NF-10 protein, we could show the existence of the NF-10 protein in all xeroderma pigmentosum complementation group cell lines. This result suggests us that NF-10 is a novel protein different from XPA protein and UV-DNA binding factor previously reported. The immunodepletion of NF-10 protein from cell extract by the antibody decreased the in vitro repair activity, whereas the additon of the protein recovered it.The partial peptide sequences of the protein are almost identical to human glyceraldehyde-3-phosphate dehydrogenase (GAPD). The purified NF-10 protein exerted the GAPD activuty in glycolysis. These results suggest that the GAPD may play a function in nucleotide excision repair as well as in glycolysis, probably in the damage recognition process.