Kalejta R F, Hamlin J L
Department of Biochemistry and Cell and Molecular Biology Program, University of Virginia School of Medicine, Charlottesville, Virginia, 22908, USA.
Exp Cell Res. 1997 Feb 25;231(1):173-83. doi: 10.1006/excr.1996.3444.
The plant amino acid, mimosine, is an extremely effective inhibitor of DNA replication in mammalian cells, but the mechanism by which this inhibition is achieved is unknown. The drug has been proposed either to inhibit initiation at origins of replication or to inhibit chain elongation by lowering nucleotide pool levels. In an attempt to determine which mode of action is correct, we have analyzed its effects on SV40 DNA replication. Using a two-dimensional gel replicon mapping technique, we show that mimosine completely inhibits incorporation of [3H]thymidine into viral DNA, but only after approximately 4 h. Qualitative analysis of replication intermediates during this interval suggests that the drug partially inhibits both initiation and elongation, and pulse-chase experiments support this contention. The drug has no effect when added directly to an SV40 in vitro replication extract. However, extracts prepared from cells pretreated with mimosine are compromised in their ability to support replication in vitro in the presence of a full complement of nucleotides. Thus, although mimosine may alter nucleotide pool levels in vivo, it also appears to affect one or more essential replication proteins.
植物氨基酸含羞草素是哺乳动物细胞中DNA复制的一种极其有效的抑制剂，但其实现这种抑制作用的机制尚不清楚。有人提出该药物要么抑制复制起点处的起始过程，要么通过降低核苷酸库水平来抑制链延长。为了确定哪种作用模式是正确的，我们分析了它对SV40 DNA复制的影响。使用二维凝胶复制子图谱技术，我们发现含羞草素完全抑制了[3H]胸苷掺入病毒DNA，但大约在4小时后才会出现这种情况。在此期间对复制中间体的定性分析表明，该药物对起始和延长过程都有部分抑制作用，脉冲追踪实验支持了这一观点。将该药物直接添加到SV40体外复制提取物中没有效果。然而，用含羞草素预处理过的细胞制备的提取物，在存在完整核苷酸的情况下，其支持体外复制的能力受到损害。因此，尽管含羞草素可能在体内改变核苷酸库水平，但它似乎也会影响一种或多种必需的复制蛋白。
