The broad goals of our work are to identify the structures which define a mammalian DNA replication origin, and to understand the mechanism of origin activation. The focus of our experiments is the replication origin of the human c-myc gene. The c-myc origin is one of a limited number of chromosomal origins identified in complex eucaryotes, and the only metazoan origin to initiate autonomous replication in vitro and in transfected cells at the chromosomal initiation sites. c-myc is an immediate early response gene, activated by many mitogenic pathways in normal cells. Multiple promoter and enhancer elements are located in the region of the c-myc replication origin, 5' to the c-myc gene. Depending on growth conditions the c-myc protein can stimulate DNA synthesis and cell division or promote apoptosis, whereas aberrant c-myc gene expression can contribute to oncogenesis. These observations suggest that the c-myc replication origin region may be an intersection point for cellular networks of transcription and growth control. As such, the c-myc origin may be a site of action of oncogenic viral or chemical pathogens. Understanding the function of DNA elements in the c-myc replication origin is likely therefore to give new insight into mechanisms which control the cell division in normal and pathological states.
Three Specific Aims will test the hypothesis that the c-myc origin comprises start sites for DNA synthesis, and cis-acting origin elements which regulate replication initiation. c-myc origin mutants will be constructed which have site-specific deletions or DNA substitutions.
Aim 1 will examine the replication of the c-myc origin constructs targeted to specific chromosomal integration sites by adeno-associated virus vectors or the yeast FLP recombines. To contrast the activity of the c-myc origin in plasmids and in the chromosome, Aim 2 will use these constructs to identify sequences which affect the ability of plasmids to replicate autonomously in 293S cell extracts, and in transfected HeLa cells.
These Aims will test the relative efficiencies of the origin constructs, and map the locations of initiation events. Also in Aims 1 and 2, nuclease digestion will be used to probe the chromatin structure of the active and inactive origin constructs.
Aim 3 will identify protein binding sites in elements which influence the activity of the c-myc replication origin. c-myc origin fragments identified by mutation as important for plasmid ARS activity or chromosomal origin activity, and supercoiled c-myc origin constructs, will be incubated with 293S cell extracts in vitro under replication initiation conditions. Specific protein:DNA complexes will be analyzed by electrophoretic mobility shift and DNase I footprinting.
