In the Chinese hamster dihydrofolate reductase replication initiation zone, the ori-beta locus is preferred over other start sites. To test the hypothesis that ori-beta contains a genetic replicator, we restored a deletion in the 3' end of the DHFR gene with a cosmid that provides the missing sequence and simultaneously knocks out the downstream ori-beta locus. Replication initiates normally in ori-beta knockout cell lines, and the DHFR domain is still synthesized in early S phase. However, initiation is completely suppressed in the starting deletion variant lacking the 3' end of the gene. We conclude that ori-beta does not contain an essential replicator, but that distant sequence elements have profound effects on origin activity in this locus.

The plant amino acid, mimosine, is an extremely effective inhibitor of DNA replication in mammalian cells (Mosca, P. J., Dijkwel, P. A., and Hamlin, J. L. (1992) Mol. Cell. Biol. 12, 4375-4383). Mimosine appears to prevent the formation of replication forks at early-firing origins when delivered to mammalian cells approaching the G 1 /S boundary, and blocks DNA replication when added to S phase cells after a lag of ϳ2.5 h. We have shown previously that [ 3 H]mimosine can be specifically photocross-linked both in vivo and in vitro to a 50-kDa polypeptide (p50) in Chinese hamster ovary (CHO) cells. In the present study, six tryptic peptides (58 residues total) from p50 were sequenced by tandem mass spectrometry and their sequences were found to be at least 77.5% identical and 96.5% similar to sequences in rabbit mitochondrial serine hydroxymethyltransferase (mSHMT). This assignment was verified by precipitating the [ 3 H]mimosine-p50 complex with a polyclonal antibody to rabbit cSHMT. The 50-kDa cross-linked product was almost undetectable in a mimosine-resistant CHO cell line and in a CHO gly ؊ cell line that lacks mitochondrial, but not cytosolic, SHMT activity. The gly ؊ cell line is still sensitive to mimosine, suggesting that the drug may inhibit both the mitochondrial and the cytosolic forms. SHMT is involved in the penultimate step of thymidylate biosynthesis in mammalian cells and, as such, is a potential target for chemotherapy in the treatment of cancer.Our laboratory's interest is the regulation of DNA synthesis in mammalian cells and, in particular, the nature of origins of replication. Although it is known that mammalian DNA is replicated from bidirectional origins spaced ϳ100 kilobase pairs apart (1), the molecular mechanisms of this process remain elusive (see Ref. 2, for review).In the absence of a viable assay for identifying the genetic elements (replicators) that control initiation in mammalian cells, attention has been focussed on determining the positions at which replication initiates, which should lie close to replicators. This approach requires methods for obtaining cell populations in which initiation at a given origin is occurring at the same time. In a commonly used synchronization protocol, cells are first arrested in the G 0 (non-proliferating) compartment by nutritional or serum starvation, followed by release into an inhibitor of DNA synthesis (e.g. Refs. 3-5). The drug treatment is enforced for a time long enough to allow all cells in the population to arrive at the beginning of the S period (a time when at least some origins are sure to be firing); the drug is then removed, allowing cells to enter S in a semi-synchronous wave. Unfortunately, this protocol is not entirely satisfactory for examining initiation events at the beginning of S, because even the most efficacious replication inhibitors do not inhibit initiation per se; rather, they slow the rate of replication fork movement by affecting DNA polymerases (e.g. aphidicolin (6)) or by lowering deoxyribonucleotide pools (e.g. hy...

Using neutral/neutral and neutral/alkaline two-dimensional (2-D) gel techniques, we previously obtained evidence that initiation can occur at any of a large number of sites distributed throughout a broad initiation zone in the dihydrofolate reductase (DHFR) domain of Chinese hamster ovary (CHO) cells. However, other techniques have suggested a much more circumscribed mode of initiation in this locus. This dichotomy has raised the issue whether the patterns of replicating DNA on 2-D gels have been misinterpreted and, in some cases, may represent such noncanonical replication intermediates as broken bubbles or microbubbles. In an accompanying study (R. F. Kalejta and J. L. Hamlin, Mol. Cell. Biol. 16:4915-4922, 1996), we have shown that broken bubbles migrate to unique positions in three different gel systems and therefore are not likely to be confused with classic replication intermediates. Here, we have applied a broken bubble assay developed from that study to an analysis of the amplified DHFR locus in CHO cells. This assay gives information about the number and positions of initiation sites within a fragment. In addition, we have analyzed the DHFR locus by a novel stop-and-go-alkaline gel technique that measures the size of nascent strands at all positions along each arc in a neutral/neutral 2-D gel. Results of these analyses support the view that the 2-D gel patterns previously assigned to classic, intact replication bubbles and single-forked structures indeed correspond to these entities. Furthermore, potential nascent-strand start sites appear to be distributed at very frequent intervals along the template in the intergenic region in the DHFR domain.Because of the great complexity of higher eukaryotic genomes, the identification of origins of replication has proven to be extremely challenging. In the absence of a reliable genetic assay for identifying autonomously replicating sequence elements, several origin mapping strategies have been devised to localize start sites in a region of interest, with the assumption that the responsible genetic element (replicator) will lie close by. The Chinese hamster dihydrofolate reductase (DHFR) replicon (Fig. 1A) has been analyzed by almost every available replicon mapping method, and all methods suggest that an origin lies somewhere in the 55-kb spacer region between the DHFR and 2BE2121 genes (reviewed in references 8, 13, and 25). However, different methods of analysis have not provided an entirely coherent picture of initiation in this locus. For example, results from high-resolution intrinsic labelling in the early S period (1, 9, 23, 32), leading-and lagging-strand polarities (10, 24, 26), and nascent-strand size and/or abundance measurements (44) are all compatible with two preferred sites or zones of initiation (termed ori-␤ and ori-␥ [32]) that are separated by about 22 kb and that lie in the 55-kb spacer region between the DHFR and 2BE2121 genes (Fig. 1A) (1, 32). In contrast, both neutral/neutral (5) and neutral/alkaline (40) two-dimensional (2-D) gel replicon...

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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.