ENZYMOLOGY OF EUCARYOTIC DNA REPLICATION

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The objective of the proposed work is the elucidation of the mechanism(s) of DNA replication in the animal Drosophila melanogaster. This may be accomplished when purified enzymes are reconstituted in vitro to yield replication complexes which initiate and sustain DNA synthesis on specific Drosophila DNA sequences. A combined biochemical and genetic approach will be employed to obtain, characterize, fractionate, and reconstitute cell-free extracts of embryos of Drosophila melanogaster that specifically replicate recombinant DNAs containing Drosophila chromosomal and mitochondrial DNA sequences. This will be accomplished by: (i) molecular cloning and characterization of origins of replication from Drosophila melanogaster; (ii) preparing and examining embryo extracts for their ability to initiate and sustain DNA replication; and (iii) purifying enzymes implicated in the DNA synthesis reactions in order to study their mechanisms of action. The control of animal cell reproduction during normal development, and the loss of control during cancerous development, is of central importance in the processes of human growth, aging, and disease. Prior to every cell division, the DNA complement of each cell is duplicated. Because DNA replication and cell division are tightly coupled, an understanding of the mechanism of DNA replication in animal cells is essential to our understanding of these basic processes.