DNA replication is essential for cell proliferation, and is under strict surveillance since abnormal replication can lead to genomic instability, tumor formation or apoptosis. Drugs that target the process of replication are central to cancer chemotherapy, and insight into the control of replication may lead to new treatment strategies. The primary cell cycle control of replication occurs at replicators and replication origins, where the initiation of DNA synthesis depends on the assembly of multiprotein complexes. The human c-myc replicator will be used as a model system. We previously identified the c-myc replicator and demonstrated its activity at the endogenous c-myc locus, in transfected plasmids in human cells, in plasmids replicated in vitro, and at an ectopic site in the HeLa genome. Recently, we used the yeast FLP recombinase to target mutagenized c-myc replicator constructs to a specific genomic site and identify sequences essential for origin activity. Using one of these sequences as bait in a yeast one-hybrid assay we discovered the c-myc DNA unwinding element binding protein DUE-B. We will continue to use the FLP recombinase system to generate clonal human cell lines with targeted integration of c-myc replicator constructs. We will test several aspects of a model in which epigenetic factors specify replication initiation sites and the DUE-B protein is involved in the loading of DNA repair proteins at presumptive replication forks. Aim 1 will alter the structure of the c-myc replicator by methylation or substitution mutagenesis; Aim 2 will assess the relationship between protein binding and replicator activity. All replicator mutants will be analyzed at the same chromosomal acceptor site. Replicator activity and structure will be analyzed by quantitative PCR, DNase digestion, and chromatin immunoprecipitation. In Aim 3 we will characterize the DUE-B protein and its role in DNA replication using physical, biochemical and immunological methods.