The process of DNA replication is the primary physiological target for anti-proliferative drugs used to treat cancer. The broad goal of our work is to characterize the major cell cycle regulated step in mammalian DNA replication, the activation of origins to initiate DNA synthesis. Our experiments focus on the human c-myc replication origin. Understand the function of DNA elements in the c-myc origin is likely to give new insight into the regulation of DNA metabolism by mechanisms that control cell division in normal and disease states. The c-myc origin is one of a limited number of chromosomal origins identified in metazoans, and the only origin to replicate autonomously in plasmids in transfected cells and in vitro at chromosomal initiation sites. To test the effects of mutations on c-myc origin activity in the chromosomes of intact cells, we have developed an innovative system based on the S. cerevisae FLP recombinase for the site-specific integration of DNA in human cells. This system is highly efficient, and reproducibly targets c-myc origin constructs with precision to specific genomic acceptor sites. The FLP recombinase system is extremely flexible in the range of constructs that can be tested in an in vivo chromosomal environment. Hence, the system is not limited to the analysis of DNA replication but is broadly applicable to the study of other aspects of DNA metabolism. Using the FLP system we will test the hypothesis that the c-myc origin compromises preferred start sites for DNA synthesis and that initiation at these sites depends on cis-acting replicator elements. In each of three Specific Aims a panel of c-myc origin constructs will be integrated at defined chromosomal acceptor sites and the structure and replication activity at those sites before and after integration of the wild type and mutated origins will be assessed.
Aim 1 will creative progressive 5' or 3' deletions of the origin, and mutations in specific candidate replicator elements, for analysis of origin activity.
Aim 2 will test directly whether c-myc origin activity or replication timing is affected by an active transcription unit or telomere position effects.
Aim 3 will test whether replication timing is affected by an active transcription unit or telomere position affects.
Aim 3 will test whether there are multiple preferred start sites for the initiation of DNA synthesis in the c-myc origin that are subservient to a cis-acting replicator. Origin activity and structure will be analyzed by competitive PCR, PCR mapping of nascent DNA strands, DNase digestion, chemical footprinting, and ligation-mediated PCR.
