Weisman-Shomer P, Fry M
Unit of Biochemistry, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa.
J Biol Chem. 1993 Feb 15;268(5):3306-12.
The single-stranded oligomer Q, whose nucleotide sequence 5'-d(TACAGGGGAGCTGGGGTAGA)-3' corresponds to the IgG switch region, forms in concentrated solutions and in the presence of alkali metal cation parallel four-stranded complexes termed G4 DNA (Sen, D., and Gilbert, W. (1988) Nature 334, 364-366). We show that G4 DNA was also formed during storage of dried oligomer Q. This quadruplex complex migrated more slowly than mono-strand oligomer Q during nondenaturing gel electrophoresis, the rate of its formation depended on the mass of stored oligomer Q, and N7 positions of guanine residues were involved in its stabilization. Here we report the purification of a protein designated QUAD that binds specifically to the G4 form of oligomer Q, from non-histone protein extracts of rabbit hepatocytes. QUAD was 80-90% purified by sequential steps of column chromatography on Sepharose 6B, DEAE-cellulose, phosphocellulose, and phenyl-Sepharose. Purified QUAD migrated on SDS-polyacrylamide gel electrophoresis as a 58 +/- 2.6-kDa polypeptide and had a native molecular mass of 57 +/- 2.5 kDa as determined by Sepharose 6B gel filtration. The dissociation constant of G4 DNA binding to QUAD was in the range of 2.5 to 7.0 x 10(-9) M/liter. Excess unlabeled monostranded oligomer Q did not compete with 5'-32P-labeled G4 DNA on its binding to QUAD. Further, that QUAD recognized the G4 DNA structure rather than a DNA sequence was also demonstrated by the inefficient competition on the binding of 5'-[32P]G4 DNA to QUAD by excess unlabeled single- or double-stranded DNA molecules that contained guanine clusters of different length or various other nucleotide sequences.
单链寡聚物Q的核苷酸序列5'-d(TACAGGGGAGCTGGGGTAGA)-3'与IgG转换区相对应，在浓溶液中且在碱金属阳离子存在下会形成称为G4 DNA的平行四链复合物（森，D.，和吉尔伯特，W.（1988年）《自然》334卷，364 - 366页）。我们发现干燥的寡聚物Q在储存过程中也会形成G4 DNA。在非变性凝胶电泳中，这种四链复合物的迁移速度比单链寡聚物Q慢，其形成速率取决于储存的寡聚物Q的质量，并且鸟嘌呤残基的N7位置参与其稳定化。在此我们报告从兔肝细胞的非组蛋白提取物中纯化出一种名为QUAD的蛋白质，它能特异性结合寡聚物Q的G4形式。通过在琼脂糖6B、DEAE - 纤维素、磷酸纤维素和苯基 - 琼脂糖上进行柱色谱的连续步骤，QUAD被纯化了80 - 90%。纯化后的QUAD在SDS - 聚丙烯酰胺凝胶电泳上作为一条58±2.6 kDa的多肽迁移，通过琼脂糖6B凝胶过滤测定其天然分子量为57±2.5 kDa。G4 DNA与QUAD结合的解离常数在2.5至7.0×10⁻⁹摩尔/升范围内。过量未标记的单链寡聚物Q在其与QUAD的结合上不能与5'-32P标记的G4 DNA竞争。此外，过量未标记的含有不同长度鸟嘌呤簇或各种其他核苷酸序列的单链或双链DNA分子对5'-[32P]G4 DNA与QUAD结合的竞争效率低下，这也证明了QUAD识别的是G4 DNA结构而非DNA序列。
