The structure of replication origins in metazoans is only nominally similar to that in model organisms, such as Saccharomyces cerevisiae. By contrast to the compact origins of budding yeast, in metazoans multiple elements act as replication start sites or control replication efficiency. We first reported that replication forks diverge from an origin 5 to the human c-myc gene and that a 2.4-kb core fragment of the origin displays autonomous replicating sequence activity in plasmids and replicator activity at an ectopic chromosomal site. Here we have used clonal HeLa cell lines containing mutated c-myc origin constructs integrated at the same chromosomal location to identify elements important for DNA replication. Replication activity was measured before or after integration of the wild-type or mutated origins using PCR-based nascent DNA abundance assays. We find that deletions of several segments of the c-myc origin, including the DNA unwinding element and transcription factor binding sites, substantially reduced replicator activity, whereas deletion of the c-myc promoter P 1 had only a modest effect. Substitution mutagenesis indicated that the sequence of the DNA unwinding element, rather than the spacing of flanking sequences, is critical. These results identify multiple functional elements essential for c-myc replicator activity.The initiation of eukaryotic DNA synthesis is precisely regulated both at the G 1 /S phase checkpoint of the mitotic cycle and by mechanisms that control the order of replicon firing (reviewed in references 9, 18, and 21). For Escherichia coli, mammalian viruses, and the budding yeast Saccharomyces cerevisiae the initiation of DNA replication is controlled by transacting initiator proteins that interact with cis-acting DNA replicator sequences. For S. cerevisiae, replicators encompass 100 to 200 bp and include the major replication origin sites where DNA synthesis begins (8). These replicators contain an essential ϳ11-bp autonomous replicating sequence (ARS) consensus sequence (ACS) that binds the origin recognition complex (ORC) to nucleate formation of prereplication complexes. By comparison, the domains that control replication in the fission yeast Schizosaccharomyces pombe are 500 to 1,500 bp in size and comprise multiple regions that add synergistically to origin activity (11,22,28,48). The sequences that contribute to origin activity in S. pombe are heterogeneous, although AϩT-rich potential binding sites for spORC have been found in several origins (10,30,36). Hence, despite notable differences in the structures of their respective ORC orthologs, the dispersed replication origins of metazoan cells more closely resemble those of S. pombe than those of S. cerevisiae in that multiple elements distributed over large distances act as replication start sites or control replication (3,4,13,15,16,27,40,53,56,58,62).Replication begins in the 5Ј flanking region of the human c-myc gene, and a 2.4-kb core fragment of the c-myc origin displays plasmid ARS activity in transfected cells and in...

No abstract

In Escherichia coli, DNA methylation regulates both origin usage and the time required to reassemble prereplication complexes at replication origins. In mammals, at least three replication origins are associated with a high density cluster of methylated CpG dinucleotides, and others whose methylation status has not yet been characterized have the potential to exhibit a similar DNA methylation pattern. One of these origins is found within the ϳ2-kilobase pair region upstream of the human c-myc gene that contains 86 CpGs. Application of the bisulfite method for detecting 5-methylcytosines at specific DNA sequences revealed that this region was not methylated in either total genomic DNA or newly synthesized DNA. Therefore, DNA methylation is not a universal component of mammalian replication origins. To determine whether or not DNA methylation plays a role in regulating the activity of origins that are methylated, the rate of remethylation and the effect of hypomethylation were determined at origin ␤ (ori-␤), downstream of the hamster DHFR gene. Remethylation at ori-␤ did not begin until ϳ500 base pairs of DNA was synthesized, but it was then completed by the time that 4 kilobase pairs of DNA was synthesized (<3 min after release into S phase). Thus, DNA methylation cannot play a significant role in regulating reassembly of prereplication complexes in mammalian cells, as it does in E. coli. To determine whether or not DNA methylation plays any role in origin activity, hypomethylated hamster cells were examined for ori-␤ activity. Cells that were >50% reduced in methylation at ori-␤ no longer selectively activated ori-␤. Therefore, at some loci, DNA methylation either directly or indirectly determines where replication begins.In early embryos undergoing rapid cell cleavage (e.g. frogs, flies, sea urchin, fish), initiation of DNA replication appears neither to require specific DNA sequences nor to occur at specific DNA sites. However, as development progresses past the blastula stage, initiation of DNA replication begins to occur at specific sites (1, 2). Thus, it is not surprising that initiation sites for DNA replication in cultured mammalian cells occur at specific genomic loci (3). For example, a 200-kb 1 region at the human ␤-globin gene (4, 5) and a 500-kb region at the mouse IgH gene (6) are both replicated from a single initiation locus. Moreover, what previously had been viewed as random initiation events distributed throughout "initiation zones" in Schizosaccharomyces pombe and hamster cells more likely reflects the presence of several strongly preferred initiation sites (7,8).Nevertheless, the precise size and composition of these initiation loci, as well as the parameters that define them remain the subject of intense investigation.The fact that specific sites for initiation of DNA replication can be developmentally acquired makes it clear that initiation sites in the metazoa are determined at least in part by epigenetic parameters. These parameters include nuclear structure, chromatin structure, and even...

DNA replication starts at multiple discrete sites across the human chromosomal c-myc region, including two or more sites within 2.4 kb upstream of the c-myc gene. The corresponding 2.4-kb c-myc origin fragment confers autonomously replicating sequence (ARS) activity on plasmids, which specifically initiate replication in the origin fragment in vitro and in vivo. To test whether the region that displays plasmid replicator activity also acts as a chromosomal replicator, HeLa cell sublines that each contain a single copy of the Saccharomyces cerevisiae FLP recombinase target (FRT) sequence flanked by selectable markers were constructed. A clonal line containing a single unrearranged copy of the transduced c-myc origin was produced by cotransfecting a donor plasmid containing the 2.4-kb c-myc origin fragment and FRT, along with a plasmid expressing the yeast FLP recombinase, into cells containing a chromosomal FRT acceptor site. The amount of short nascent DNA strands at the chromosomal acceptor site was quantitated before and after targeted integration of the origin fragment. Competitive PCR quantitation showed that the c-myc origin construct substantially increased the amount of nascent DNA relative to that at the unoccupied acceptor site and to that after the insertion of non-myc DNA. The abundance of nascent strands was greatest close to the c-myc insert of the integrated donor plasmid, and significant increases in nascent strand abundance were observed at sites flanking the insertion. These results provide biochemical and genetic evidence for the existence of chromosomal replicators in metazoan cells and are consistent with the presence of chromosomal replicator activity in the 2.4-kb region of c-myc origin DNA.The bacterial replicon model of Jacob et al. (17) proposed that a trans-acting initiator protein binds to a cis-acting replicator element, defined as "a specific element of recognition upon which the corresponding initiator would act, allowing the replication of the DNA attached to the replicator." Consistent with this model, replication initiation in the simple eucaryote Saccharomyces cerevisiae is regulated by the interaction of cell cycle-dependent trans-acting factors with cis-acting DNA elements to establish replication bubbles and cause the initiation of DNA synthesis (6, 36). In metazoans, however, putative cis-acting replicator elements may be distributed over larger distances than the compact replicators of S. cerevisiae (4, 37), effect initiation at multiple sites, and comprise features of nuclear, chromatin, or DNA structure as well as DNA sequence (6,8,14,15). Biochemical assays suggest that DNA synthesis can initiate at multiple sites over regions as large as 55 kb in the hamster dihydrofolate reductase (DHFR) locus (9), the human c-myc locus (39, 41), and elsewhere (2, 7, 24, 35, 42). Replication does not initiate randomly across these large zones, although the degree to which replication initiates at preferred sites varies between replicons (13, 18, 20, 32). Nevertheless, the start sites f...

Clean and spiked sediment formulations of various silt sand and clay sand ratios were tested for toxicity using a bioassay that utilizes bioluminescent bacteria Measured toxicities of clean and copper sulfate-spiked sediments were negatively but nonlinearly related with percent silt and percent clay, but no significant relationship existed between measured toxicity and sediment composition for methyl parathion-spiked formulations Results suggest that solid phase sediment bioassays using bio luminescent bacteria may be useful for testing the toxicities of single contaminants in formulated artificial sediments of known particle size composition, and for repeated samples collected from the same site However, extreme caution must be taken when testing sediments of varying composition or which may be differentially contaminated or contain a suite of contaminants