Mimosine, a novel inhibitor of DNA replication, binds to a 50 kDa protein in Chinese hamster cells

Abstract

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.

Mimosine Targets Serine Hydroxymethyltransferase

“…Furthermore, we demonstrated that the p50 mimosine binding activity is almost absent in a CHO cell line selected for resistance to 1 mM mimosine (ϳ10 times the lethal dose; 19,20). p50 partitions largely with the soluble cytoplasmic and nuclear fractions, and the ability to bind mimosine does not fluctuate demonstrably during the cell cycle (19).…”

“…We recently obtained evidence for a different (or additional) intracellular target. We showed that [ 3 H]mimosine can be specifically cross-linked to a 50-kDa polypeptide (p50) both in vivo and in vitro at concentrations equal to the minimum effective dose in vivo (19,20). Furthermore, we demonstrated that the p50 mimosine binding activity is almost absent in a CHO cell line selected for resistance to 1 mM mimosine (ϳ10 times the lethal dose; 19,20).…”

“…In addition, it has no apparent effect on DNA synthesis either in frog embryos (18) or in in vitro replication extracts prepared from mammalian cells (16,17), 2 both of which contain large deoxynucleotide pools. Furthermore, the inhibitory effects of mimosine on DNA replication in CHO cells can be overcome by adding iron to the culture medium (19). It has therefore been suggested that mimosine functions solely by inhibiting ribonucleotide reductase (16,17).…”

“…19). However, a simple lowering of deoxyribonucleotide pool levels by inhibiting ribonucleotide reductase does not explain why mimosine is such an efficacious inhibitor and why it appears to prevent initiation of nascent DNA chains.…”

“…We showed that [ 3 H]mimosine can be specifically cross-linked to a 50-kDa polypeptide (p50) both in vivo and in vitro at concentrations equal to the minimum effective dose in vivo (19,20). Furthermore, we demonstrated that the p50 mimosine binding activity is almost absent in a CHO cell line selected for resistance to 1 mM mimosine (ϳ10 times the lethal dose; 19,20). p50 partitions largely with the soluble cytoplasmic and nuclear fractions, and the ability to bind mimosine does not fluctuate demonstrably during the cell cycle (19).…”