Research at the DNA replication group aims at understanding how information from the cell cycle machinery leads to the initiation of DNA replication. Proper cell growth depends on a network of interacting molecules that prevents DNA replication and cell division under unfavorable conditions. Disruptions in the intricate balance between components of this network may lead to cancer; however, interfering with signals transmitted by the cell cycle signaling network is an important tool for cancer therapy. A better understanding of the cell cycle is fundamental to the development of rational, knowledge-based strategies to combat cancer and utilize stem cells to improve human health. To study cell cycle signaling at the chromatin level, we specify DNA sequences that determine whether, where, and when replication will occur. DNA sequences that determine the location of replication initiation are called replicators. Replicators are identified by their ability to start replication when transferred from their original genomic locus to ectopic genomic sites. Genetic dissection of replicators allows us to delineate the sequence requirements for starting DNA replication. We had also reported that the timing of DNA replication during the S-phase of the cell cycle can be altered, and are now elucidating the genetic and epigenetic factors that determine replication timing. Understanding replicators and replication timers will eventually lead to the identification of proteins that interact with chromatin to exert cell cycle control of DNA replication. We perform these analyses in established cell lines and in embryonic or hematopoietic stem cells. We use the beta-globin locus as a model system to study the determination of replication sites and timing in mammalian cells. In human cells, replication starts from a region termed IR, located between the two adult beta globin genes. Our studies demonstrated that IR can function as a replicator, facilitating initiation of DNA replication when transferred to an ectopic location (2). On the other hand, we have shown that in mouse cells, DNA replication initiates from multiple sites within the beta-globin locus (3). Nevertheless, the replication timing pattern remains similar to that observed in human cells - cells that do not express globin replicate the locus late during S-phase whereas cells that express the protein replicate the locus early during S-phase. This study had also shown that murine embryonic stem cells (ES cells) initiated DNA replication at specific sites, unlike some other embryonic systems in which replication initiation appeared random. This was especially interesting because we have previously observed that ES cells do not activate some of the cell cycle controls that regulate the growth of normal somatic cells (4). We now use the murine system in an attempt to understand the interrelations between the timing of DNA replication, gene expression and cell cycle control. The studies described above demonstrated that replication at the human beta globin locus requires specific DNA sequences. Next, we asked which sequences within IR dictate replicator activity. We found that there are two independent replicators within the globin IR (9). Each one of these replicators can act as a DNA replication starting site. Within each replicator, we identified two sequences that cooperate to facilitate replication. Recently we have demonstrated that sequences essential for initiation of DNA replication in one of the replicators could cooperate with sequences from the other replicator to initiate replication (10). These studies suggest that replicators are modular and that the location of initiation sites can be determined cooperatively by several combinations of essential sequences. We have also found that one of the replicator sequences binds a protein during the early stages of the cell cycle; deletion of the protein binding sequence prevents initiation of DNA replication.
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