Keeney S, Chang G J, Linn S
Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.
J Biol Chem. 1993 Oct 5;268(28):21293-300.
A human DNA damage binding protein implicated in the DNA excision repair disorder xeroderma pigmentosum E was purified to near homogeneity from HeLa cells. The protein is abundant (approximately 10(5) copies/cell) and has a native molecular weight of 154,000-163,000 as estimated by gel filtration and glycerol gradient sedimentation. DNA damage binding activity copurified with polypeptides of 124 and 41 kDa. Based on the native molecular weight, cosedimentation of both polypeptides with DNA damage binding activity on glycerol gradients, and a molar ratio of approximately 1:1 for the two polypeptides, it appears that p124 and p41 are subunits of a heterodimeric protein. Binding to damaged DNA was resistant to K+ concentrations approaching 1 M, but showed anion-specific sensitivity to Cl- concentrations above 0.5 M, suggesting that the majority of the binding energy is contributed by nonionic interactions. In contrast to previous reports, the DNA damage binding protein was shown to recognize cyclobutane pyrimidine dimers in addition to a nonphotoreactivable lesion(s), most likely the pyrimidine-pyrimidone (6-4) photoproduct.
一种与DNA切除修复障碍性疾病着色性干皮病E相关的人类DNA损伤结合蛋白从HeLa细胞中纯化至接近均一。该蛋白含量丰富（约10⁵个拷贝/细胞），通过凝胶过滤和甘油梯度沉降估计其天然分子量为154,000 - 163,000。DNA损伤结合活性与124 kDa和41 kDa的多肽共纯化。基于天然分子量、两种多肽在甘油梯度上与DNA损伤结合活性的共沉降以及两种多肽约1:1的摩尔比，似乎p124和p41是一种异二聚体蛋白的亚基。与受损DNA的结合对接近1 M的K⁺浓度具有抗性，但对高于0.5 M的Cl⁻浓度表现出阴离子特异性敏感性，这表明大部分结合能量由非离子相互作用贡献。与先前的报道相反，该DNA损伤结合蛋白除了能识别一种不可光复活的损伤（最可能是嘧啶 - 嘧啶酮(6 - 4)光产物）外，还能识别环丁烷嘧啶二聚体。
