IN-VITRO REPLICATION OF PLASMIDS CONTAINING HUMAN C-MYC DNA

A chromosomal replication initiation zone was previously mapped in cel l cultures to the 5' flanking DNA of the human c-myc gene. We have use d an in vitro system to examine the replication of a plasmid, pNeo.Myc -2.4, containing 2.4 kb of the c-myc initiation zone. In vitro, pNeo.M yc-2.4 generated high levels of DpnI-resistant DNA above background in corporation into control plasmids. pNeo.Myc-2.4 replicated semiconserv atively to produce supercoiled and relaxed plasmid monomers, and repli cative intermediates. [P-32]dCMP incorporated into pNeo.Myc-2.4 appear ed in Okazaki fragments and low molecular weight strands which matured to full length plasmid DNA, whereas [P-32]dCMP incorporated into cont rol plasmids appeared as continuous smears on denaturing gels. Other a ssays also distinguished the processive replication of pNeo.Myc-2.4 fr om the dispersive labeling of control plasmids. A pNeo.Myc-2.4 replica tion time course showed a clear preference for initiation within a res triction fragment containing the c-myc DNA. Two-dimensional electropho resis revealed that a restriction fragment bearing the c-myc origin zo ne generated an are characteristic of replicative intermediates contai ning a central replication bubble, while vector fragments in the plasm id generated arcs of forked intermediates. Replication bubbles visuali zed by electron microscopy were centered within the replication initia tion zone, approximately 1.4 kb upstream of c-myc promoter P1. Okazaki fragments radiolabeled during in vitro replication showed a switch in the asymmetry of template preference within the initiation zone ident ified by electron microscopy, two-dimensional electrophoresis and earl y labeling. These data show that bidirectional, semiconservative repli cation can originate preferentially in vitro in the 5' flanking DNA of the c-myc gene, and that replicative intermediates present at low lev els can be distinguished from molecules generated by competing, repair -type processes.