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 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. Mosca et 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 functions in vivo simply 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 interaction in vitro, probably explaining why both metal ions are able to overcome mimosine's inhibitory effect on DNA synthesis in vivo.
Mosca
P J
PJ
Biophysics Program, University of Virginia School of Medicine, Charlottesville 22908.
Lin
H B
HB
Hamlin
J L
JL
D000818:Animals; D016466:CHO Cells / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002453:Cell Cycle; D003300:Copper / Q000494:pharmacology; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004261:DNA Replication / Q000187:drug effects*; D007501:Iron / Q000494:pharmacology; D008898:Mimosine / Q000378:metabolism* / Q000494:pharmacology; D008970:Molecular Weight; D010777:Photochemistry; D011506:Proteins / Q000378:metabolism*; D014316:Tritium
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D011506:Proteins; D014316:Tritium; D008898:Mimosine; D003300:Copper; D007501:Iron