DESCRIPTION: The long term objective is the elucidation of mechanisms of DNA replication in animal mitochondria. This may be accomplished, among other ways, when purified enzymes are reconstituted in vitro to yield replication complexes capable of initiation and elongation of DNA synthesis. The studies will provide the framework for research to determine the regulation of these processes, as they occur in vivo. The proposal focuses on the major replicative enzyme in Drosophila mitochondria, DNA Polymerase gamma (Pol gamma). A combined approach of biochemistry and molecular biology is being employed, to pursue mechanistic and structure-function analyses of this p125:p35 heterodimeric DNA polymerase, to overexpress and purify the two subunits both together and individually, and to elucidate the role of the small subunit in enzyme function. Additional goals are to generate altered enzyme forms for comparative studies, and to examine the role of mitochondrial single-stranded DNA-binding protein (SSB) in Pol gamma function. The applicant will also use a transgenic fly model to examine possible relationships between Pol gamma function, mitochondrial DNA replication fidelity, aging, and disease. 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. Mitochondrial biogenesis proceeds in parallel with cell proliferation, but it is neither tightly coupled to mitochondrial DNA replication nor to the cell cycle. Nevertheless, because both the DNA content of the mitochondrion and the number of mitochondria in cell remain relatively constant, specific regulatory mechanisms are likely required to couple mitochondrial DNA replication and biogenesis, to nuclear DNA replication and cell division. A detailed analysis of the key enzyme involved in mitochondrial DNA replication will represent a major contribution toward an eventual understanding of mitochondrial biogenesis and function in normal and diseased tissues. A variety of mitochondrial DNA diseases have recently been documented. This and an increased recognition that antiviral and antitumor drugs frequently affect mitochondrial DNA function, and in particular, the activity of Pol gamma demonstrate a critical need for an in-depth understanding of this DNA polymerase. The Specific Aims as follows: 1. To continue mechanistic analysis of Pol gamma to examine the mechanisms and fidelity of nucleotide polymerization, and the role of mitochondrial (mtSSB) in Pol gamma function; 2. To overexpress and purify recombinant forms of the two subunits of Pol gamma in E. coli and in bacculovirus-infected insect cells; 3. To continue structure-function studies of Pol gamma involving native, recombinant and altered forms, to elucidate the role of the small subunit in enzyme function, to dissect functional domains in the catalytic subunit, and begin to elucidate the molecular architecture of the enzyme; and 4. To initiate the development of a transgenic fly model to examine the relationship between Pol gamma function, mitochondrial DNA replication fidelity, aging and disease.