Activity of the c-myc replicator at an ectopic chromosomal location

DNA replication starts at multiple discrete sites across the human chromoso mal 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 autonom ously replicating sequence (ARS) activity on plasmids, which specifically i nitiate replication in the origin fragment in vitro and in vivo. To test wh ether the region that displays plasmid replicator activity also acts as a c hromosomal replicator, HeLa cell sublines that each contain a single copy o f the Saccharomyces cerevisiae FLP recombinase target (FRT) sequence flanke d by selectable markers were constructed. A clonal line containing a single unrearranged copy of the transduced c-myc origin was produced by cotransfe cting 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 conta ining a chromosomal FRT acceptor site. The amount of short nascent DNA stra nds at the chromosomal acceptor site was quantitated before and after targe ted integration of the origin fragment. Competitive PCR quantitation showed that the c-myc origin construct substantially increased the amount of nasc ent 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 e xistence of chromosomal replicators in metazoan cells and are consistent wi th the presence of chromosomal replicator activity in the 2.4-kb region of c-myc origin DNA.