DNA replication is an essential prelude to cell division and proliferation. The process is an important target of cell cycle control because it is critical for viability that the DNA is copied accurately and precisely at the correct time. Failure in this process generally leads to mutations within the genome. Moreover, genome stability may be compromised if DNA is replicated completely or if there is local re-initiation of replication because abnormal partitioning of the DNA would occur during mitosis. These kinds of irregularities have been found to be associated with different types of cancer. These kinds of irregularities have been found to be associated with different types of cancer. Thus, elucidating the regulation and mechanism of DNA replication would be useful for designing newer drugs to combat these diseases. At present, the origins of DNA replication or proteins that recognize them have not been identified and characterized in great detail in humans and other mammalians. To begin to distinguish these origins, we first propose to purify from human cells purify from human cells a set of proteins, the origin recognition complex (ORC), that is presumed to bind to origins. We then intend to identify the target binding sites of human ORC and characterize the specificity of the protein-DNA interactions, Lastly, we wish to clone the remaining subunits of human ORC. These studies are an important first step towards the detailed characterization of human origins of replication. More significantly, this proposal is essential groundwork for developing a human cell-free system that is capable of initiating DNA synthesis from a specific human origin, which will be crucial for experiments designed to elucidate the precise mechanism of initiation of DNA replication.
