The overall goal of this proposal is to identify DNA sequences and protein factors regulating the initiation of DNA replication in human cells. We shall capitalize on two new developments from this laboratory. Firstly, we shall exploit a new PCR-based procedure that allows rapid and sensitive mapping of initiation sites over long stretches of mammalian genomic DNA. Lung tumor cell lines offer a unique opportunity for mapping since different cell lines possess amplified chromosome regions containing each of three different members of the myc gene family. We have at this time localized a zone of initiation of replication upstream of the human c-myc gene. We shall continue to refine mapping of this zone and to identify sites of initial bidirectional replication in lung tumor cells. Using our method, we shall map origins of replication in the vicinity of N-myc and L-myc genes. We shall determine whether origins of replication are the same in cells with either unamplified or amplified myc family genes. We shall compare sequences at initiation zones for the myc family members and identify any consensus elements. Secondly, we seek to quickly capitalize on our recent discovery of the PUR element, a 16 bp consensus sequence conserved at several eukaryotic origins of replication. We have now identified a protein that binds to this element at the major DNA bending site in the c-myc initiation zone. We shall purify this protein, which has high, specific affinity for the purine-rich single strand of PUR. We shall clone and sequence the gene for this protein, a process we have already successfully begun.
Our aim i s to test the PUR-binding protein, and any other potential initiation factor, for ability to promote initiation of replication in vitro. We shall test potential regulatory factors for specific duplex-opening activity and helicase activity, two functions associated with the beginning of the initiation process. Proteins exhibiting activity will be tested in our complete in vitro replication system with cloned sequences selected for optimal efficiency. Intriguingly, PUR is present near several genes known to be amplified in their respective systems. We shall sequence the PUR element near amplified myc genes in lung tumor cell lines to see whether mutations are associated with amplification. Results will help us understand how DNA replication initiates and how this replication is aberrant at specific origins in lung cancer.
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