Todd A, Cossons N, Aitken A, Price G B, Zannis-Hadjopoulos M
McGill Cancer Centre, McGill University, Montréal, Québec, Canada.
Biochemistry. 1998 Oct 6;37(40):14317-25. doi: 10.1021/bi980768k.
Cruciform DNA has been implicated in the initiation of DNA replication. Recently, we identified and purified from human (HeLa) cells a protein, CBP, with binding specificity for cruciform DNA. We have reported previously that the CBP activity sediments at approximately 66 kDa in a glycerol gradient. Here, photochemical cross-linking studies and Southwestern analyses confirm that a 70 kDa polypeptide interacts specifically with cruciform DNA. Microsequence analysis of tryptic peptides of the 70 kDa CBP reveals that it is 100% homologous to the 14-3-3 family of proteins and shows that CBP contains the epsilon, beta, gamma, and zeta isoforms of the 14-3-3 family. In addition to polypeptides with the characteristic molecular mass of 14-3-3 proteins (30 and 33 kDa), CBP also contains a polypeptide of 35 kDa which is recognized by an antibody specific for the epsilon isoform of 14-3-3. Cruciform-specific binding activity is also detected in 14-3-3 proteins purified from sheep brain. Immunofluorescene studies confirm the presence of the epsilon, beta, and zeta isoforms of 14-3-3 proteins in the nuclei of HeLa cells. The 14-3-3 family of proteins has been implicated in cell cycle control, and members of this family have been shown to interact with various signaling proteins. Cruciform binding is a new activity associated with the 14-3-3 family.
十字形DNA与DNA复制的起始有关。最近，我们从人（HeLa）细胞中鉴定并纯化出一种对十字形DNA具有结合特异性的蛋白质CBP。我们之前报道过，CBP活性在甘油梯度中沉降于约66 kDa处。在此，光化学交联研究和蛋白质印迹分析证实，一种70 kDa的多肽与十字形DNA特异性相互作用。对70 kDa CBP的胰蛋白酶肽段进行微序列分析表明，它与14-3-3蛋白家族100%同源，且显示CBP包含14-3-3家族的ε、β、γ和ζ亚型。除了具有14-3-3蛋白特征分子量（30和33 kDa）的多肽外，CBP还包含一种35 kDa的多肽，它可被针对14-3-3 ε亚型的特异性抗体识别。从羊脑中纯化的14-3-3蛋白也检测到了十字形特异性结合活性。免疫荧光研究证实HeLa细胞核中存在14-3-3蛋白的ε、β和ζ亚型。14-3-3蛋白家族与细胞周期调控有关，且已表明该家族成员可与多种信号蛋白相互作用。十字形结合是与14-3-3家族相关的一种新活性。
