Muraiso T, Nomoto S, Yamazaki H, Mishima Y, Kominami R
First Department of Biochemistry, Niigata University School of Medicine, Japan.
Nucleic Acids Res. 1992 Dec 25;20(24):6631-5. doi: 10.1093/nar/20.24.6631.
A protein that binds to a synthetic oligonucleotide of (CCT)12 has been purified from Ehrlich ascites tumor cells by a (CCT)12 affinity chromatography. The protein (p70) has an apparent molecular mass of 70 kDa, as assayed by Southwestern analysis. A competition experiment revealed that p70 binds to (CCT)12, (CCCT)8 and (CCTCCCT)6, but not to (CTT)12, (CT)16 and (CCTGCCT)6, suggesting that p70 has a sequence-specificity. The complementary (AGG)12 and the double stranded DNA did not show the binding. It is also confirmed by S1 nuclease analysis that the (AGG:CCT)12 duplex takes a single-stranded conformation in the absence of the protein. This raises a possibility that the duplex forms two single-stranded loops in chromosomes, the C-rich strand being bound to p70. Structural analysis of the resulting (AGG)12 strand by non-denaturing polyacrylamide gel electrophoresis demonstrated the presence of slower and faster migrated conformers in a neutral pH buffer containing 50 mM NaCl at 5 degrees C. The ratio was dependent on the DNA concentration. Both conformers disappeared in the absence of NaCl. This suggests that (AGG)12 can form intra- and inter-molecular complexes by non-Watson-Crick, guanine:guanine base-pairing. The possible biological function of the (AGG:CCT)n duplex and the p70 is discussed.
一种能与（CCT）12合成寡核苷酸结合的蛋白质已通过（CCT）12亲和层析从艾氏腹水瘤细胞中纯化出来。通过蛋白质印迹分析测定，该蛋白质（p70）的表观分子量为70 kDa。竞争实验表明，p70能与（CCT）12、（CCCT）8和（CCTCCCT）6结合，但不与（CTT）12、（CT）16和（CCTGCCT）6结合，这表明p70具有序列特异性。互补的（AGG）12和双链DNA未显示出结合。S1核酸酶分析也证实，在没有该蛋白质的情况下，（AGG:CCT）12双链体呈单链构象。这增加了一种可能性，即该双链体在染色体中形成两个单链环，富含C的链与p70结合。通过非变性聚丙烯酰胺凝胶电泳对所得（AGG）12链进行结构分析，发现在5℃含有50 mM NaCl的中性pH缓冲液中存在迁移较慢和较快的构象异构体。该比例取决于DNA浓度。在没有NaCl的情况下，两种构象异构体均消失。这表明（AGG）12可通过非沃森-克里克鸟嘌呤：鸟嘌呤碱基配对形成分子内和分子间复合物。本文讨论了（AGG:CCT）n双链体和p70可能的生物学功能。
