Mimosine, a novel inhibitor of DNA replication, binds to a 50 kDa protein in Chinese hamster cells.
Mimosine, a novel inhibitor of DNA replication, binds to a 50 kDa protein in Chinese hamster cells.
复制标题
Mimosine 是一种新型 DNA 复制抑制剂，可与中国仓鼠细胞中的 50 kDa 蛋白质结合。
DOI：
10.1093/nar/23.2.261
复制
发表时间：
1995
影响因子：
14.9
通讯作者：
Hamlin,JL
中科院分区：
文献类型：
--
作者：
Mosca,PJ;Lin,HB;Hamlin,JL
我们最近证明了植物氨基酸含羞草碱是哺乳动物细胞中DNA复制的极其有效的抑制剂[P.A. Dijkwel和J. L. 03 The Dog（1992）cell. 12，3715 - 3722; P.J.Mosca等人（1992）Mol. Biol. cell. 12，4375 - 4383]。它的一些性质进一步表明，含羞草碱可能靶向复制起点的起始，这将使其成为一个独特的和非常有用的抑制剂，用于研究DNA合成的调控。然而，含羞草素已知螯合铁，核糖核苷酸还原酶的辅因子。因此，含羞草碱在体内的功能可能仅仅是通过降低细胞内的脱氧核糖核苷酸库来实现的。在本研究中，我们表明，事实上，它是可以覆盖含羞草素抑制在体内添加过量的铁，然而，铜，这是不是一个替代品的铁在核糖核苷酸还原酶，是同样有效的。有证据表明，含羞草素的功能，而不是通过结合到细胞内的蛋白质。我们表明，放射性标记的含羞草素可以在体外特异性交联到一个50 kDa的多肽（称为p50）。与p50的结合在选择对ImM含羞草碱具有抗性的CHO细胞中几乎检测不到，这表明p50是生物学相关的靶标。p50与细胞膜部分无关，因此可能不是通道蛋白。此外，结合活性不显着变化的功能，细胞周期的位置，认为p50不是一个周期蛋白。最后，铁和铜都能够在体外逆转含羞草碱与p50的相互作用，这可能解释了为什么两种金属离子都能够克服含羞草碱对体内DNA合成的抑制作用。
We recently demonstrated that the plant amino acid, mimosine, is an extremely efficacious inhibitor of DNA replication in mammalian cells [P. A. Dijkwel and J. L. Hamlin (1992)Mol. Cell. Biol. 12, 3715–3722; P. J. Moscaet al. (1992)Mol. Cell. Biol. 12, 4375–4383]. Several of its properties further suggested that mimosine might target initiation at origins of replication, which would make it a unique and very useful inhibitor for studying the regulation of DNA synthesis. However, mimosine is known to chelate iron, a cofactor for ribonucleotide reductase. Thus, the possibility arose that mimosine functionsin vivosimply by lowering intracellular deoxyribonucleotide pools. In the present study, we show that, in fact, it is possible to override mimosine inhibition in vivo by adding excess iron; however, copper, which is not a substitute for iron in ribonucleotide reductase, is equally effective. Evidence is presented that mimosine functions instead by binding to an intracellular protein. We show that radiolabeled mimosine can be specifically cross-linked to a 50 kDa polypeptide (termed p50)in vitro. Binding to p50 is virtually undetectable in CHO cells selected for resistance to 1 mM mimosine, arguing that p50 is the biologically relevant target. p50 is not associated with the cellular membrane fraction and, hence, is probably not a channel protein. Furthermore, the binding activity does not vary markedly as a function of cell cycle position, arguing that p50 is not a cyclin. Finally, both iron and copper are able to reverse the mimosine-p50 interactionin vitro, probably explaining why both metal ions are able to overcome mimosine's inhibitory effect on DNA synthesisin vivo.