The mitochondrial replicase (pol gamma) comprises two subunits, a large catalytic core (pol gamma-alpha) and a smaller accessory subunit (pol gamma-betta) that enhances holoenzyme activity and processivity. Mutagenesis of four conserved sequence elements located within the spacer region between the DNA polymerase and 3'-5' exonuclease active sites in pol gamma-alpha demonstrates their functional roles in holoenzyme activity, processivity and/ or DNA binding affinity. Several mutations also affect differentially DNA polymerase and exonuclease activity, and/ or functional interactions with mitochondrial single-stranded DNA-binding protein (mtSSB). Overexpression of the catalytic core in the nervous system of Drosophila induces severe mtDNA depletion and reduces median life span. A parallel mutagenesis of the human accessory subunit, in combination with the determination of its crystal structure and molecular modeling, elucidates its role as a novel type of processivity factor. Loss of function alleles result in mtDNA depletion and developmental lethality in Drosophila. A human pol gamma/DNA complex model was developed using the structures of the pol gamma beta dimer and the T7 DNA polymerase ternary complex, which suggests multiple regions of subunit interaction between pol gamma beta and the human catalytic core that allow it to encircle the newly synthesized double-stranded DNA, and thereby enhance DNA binding affinity and holoenzyme processivity. Functional complexes of pol gamma, mtSSB and a novel mitochondrial DNA helicase reconstitute the mitochondrial DNA replication fork. The human mtDNA helicase exists as a hexamer/ heptamer and exhibits a modular architecture that is highly similar to that of bacteriophage T7 primase-helicase and E. coli DnaB protein. Molecular analysis of active site and selected human disease alleles of the Drosophila homolog by overexpression in Schneider cells results in a dominant-negative lethal phenotype resulting from mtDNA depletion. This work was supported by NIH grant GM45295.

Dr. Kaguni completed her B.A. degree in Biology with High Honors at the University of California, San Diego in 1974 and worked as a research assistant with Professor E. P. Geiduschek from 1974-1976. She completed her Ph.D. degree in Biology in the laboratory of Professor Dan S. Ray at the University of California, Los Angeles in 1980. Her Ph.D. research focused on the study of small phage replication and on the genetic elements and protein requirements for bacterial DNA replication. She pursued a year-long NIH-sponsored postdoctoral study in 1980 in the laboratory of Professor David Clayton at Stanford University studying site-directed pausing in DNA synthesis promoted by DNA secondary structure, and continued in 1981-1984 as an American Cancer Society postdoctoral fellow in the laboratory of Professor Robert Lehman at Stanford University, where she purified and characterized the first native form of animal DNA polymerase alpha-primase, elucidating its four-subunit structure and determining the subunit association of its two enzyme activities.

Dr. Kaguni was appointed as Assistant Professor in 1984 in the Department of Biochemistry at Michigan State University, where she currently holds the title of University Distinguished Professor. There she began her studies of the enzymology and molecular genetics of mitochondrial DNA replication, using the fruit fly Drosophila as and animal model system. Her laboratory has made seminal contributions to our understanding of the structure and functional roles of the mitochondrial DNA replicase, purifying the first native form, elucidating its subunit structure, characterizing its two catalytic activities, and cloning and expressing its two subunits and elucidating their functional roles.

For more information, visit
http://www.bch.msu.edu/faculty/kagunil.htm

NIH Director's Wednesday Afternoon Lecture