The long term objective of the proposed work is the elucidation of the mechanism of DNA replication in animal mitochondria. This may be accomplished 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 current proposal focuses on the major replicative enzyme in mitochondria, DNA polymerase gamma. A combined approach involving biochemical, immunological and molecular biological methods will be employed to undertake mechanistic and structure-function analysis of the mitochonchial DNA polymerase and to done and overproduce its two subunits. Secondary goals involve isolation of a mitochondrial RNA polymerase / DNA primase and examination of the co-ordination of RNA priming and DNA synthesis on mitochondrial DNA templates. 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 mechanisms of DNA replication functioning in animal cells is essential to our understanding of these basic processes. 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 cells 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, gamma polymerase, will represent a major contribution toward an eventual understanding of mitochondrial biogenesis.
| Salminen, Tiina S; Oliveira, Marcos T; Cannino, Giuseppe et al. (2017) Mitochondrial genotype modulates mtDNA copy number and organismal phenotype in Drosophila. Mitochondrion 34:75-83 |
| Nurminen, Anssi; Farnum, Gregory A; Kaguni, Laurie S (2017) Pathogenicity in POLG syndromes: DNA polymerase gamma pathogenicity prediction server and database. BBA Clin 7:147-156 |
| Kaguni, Laurie S; Oliveira, Marcos Túlio; Tamanoi, Fuyuhiko (2016) Preface. Enzymes 39:xi |
| Gajewski, John P; Arnold, Jamie J; Salminen, Tiina S et al. (2016) Expression and Purification of Mitochondrial RNA Polymerase and Transcription Factor A from Drosophila melanogaster. Methods Mol Biol 1351:199-210 |
| Kaguni, Laurie S; Oliveira, Marcos T (2016) Structure, function and evolution of the animal mitochondrial replicative DNA helicase. Crit Rev Biochem Mol Biol 51:53-64 |
| Ciesielski, Grzegorz L; Hytönen, Vesa P; Kaguni, Laurie S (2016) Biolayer Interferometry: A Novel Method to Elucidate Protein-Protein and Protein-DNA Interactions in the Mitochondrial DNA Replisome. Methods Mol Biol 1351:223-31 |
| Ciesielski, G L; Oliveira, M T; Kaguni, L S (2016) Animal Mitochondrial DNA Replication. Enzymes 39:255-92 |
| Rosado-Ruiz, Fernando A; So, Minyoung; Kaguni, Laurie S (2016) Purification and Comparative Assay of the Human Mitochondrial Replicative DNA Helicase. Methods Mol Biol 1351:185-98 |
| Ciesielski, Grzegorz L; Rosado-Ruiz, Fernando A; Kaguni, Laurie S (2016) Purification and Comparative Assay of Human Mitochondrial Single-Stranded DNA-Binding Protein. Methods Mol Biol 1351:211-22 |
| Ciesielski, Grzegorz L; Bermek, Oya; Rosado-Ruiz, Fernando A et al. (2015) Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase ? by Organization of the Template DNA. J Biol Chem 290:28697-707 |
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