MIMOSINE, A NOVEL INHIBITOR OF DNA-REPLICATION, BINDS TO A 50-KDA PROTEIN IN CHINESE-HAMSTER CELLS

We recently demonstrated that the plant amino acid, mimosine, is an ex tremely efficacious inhibitor of DNA replication in mammalian cells [P . A. Dijkwel and J. L. Hamlin (1992) Mol. Cell. Biol. 12, 3715-3722; P . J. Mosca et al. (1992) Mol. Cell. Biol. 12, 4375-4383]. Several of i ts properties further suggested that mimosine might target initiation at origins of replication, which would make it a unique and very usefu l inhibitor for studying the regulation of DNA synthesis. However, mim osine is known to chelate iron, a cofactor for ribonucleotide reductas e. Thus, the possibility arose that mimosine functions in vivo simply by lowering intracellular deoxyribonucleotide pools. In the present st udy, we show that, in fact, it is possible to override mimosine inhibi tion in vivo by adding excess iron; however, copper, which is not a Su bstitute for iron in ribonucleotide reductase, is equally effective. E vidence is presented that mimosine functions instead by binding to an intracellular protein. We show that radiolabeled mimosine can be speci fically crosslinked to a 50 kDa polypeptide (termed p50) in vitro. Bin ding to p50 is virtually undetectable in CHO cells selected for resist ance to 1 mM mimosine, arguing that p50 is the biologically relevant t arget. p50 is not associated with the cellular membrane fraction and, hence, is probably not a channel protein. Furthermore, the binding act ivity does not vary markedly as a function of cell cycle position, arg uing that p50 is not a cyclin. Finally, both iron and copper are able to reverse the mimosine-p50 interaction in vitro, probably explaining why both metal ions are able to overcome mimosine's inhibitory effect on DNA synthesis in vivo.